Neural progenitor cells derived from embryonic stem cells

ABSTRACT

The present invention relates to undifferentiated human embryonic stem cells, methods of cultivation and propagation and production of differentiated cells. In particular it relates to the production of human ES cells capable of yielding somatic differentiated cells in vitro, as well as committed progenitor cells such as neural progenitor cells capable of giving rise to mature somatic cells including neural cells and/or glial cells and uses thereof. This invention provides methods that generate in vitro and in vivo models of controlled differentiation of ES cells towards the neural lineage. The model, and cells that are generated along the pathway of neural differentiation may be used for: the study of the cellular and molecular biology of human neural development, discovery of genes, growth factors, and differentiation factors that play a role in neural differentiation and regeneration, drug discovery and the development of screening assays for teratogenic, toxic and neuroprotective effects.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/361,976 filed on Jan. 31, 2012, which is a division of U.S. patentapplication Ser. No. 12/157,864 filed on Jun. 13, 2008, now U.S. Pat.No. 8,137,969, which is a continuation of U.S. patent application Ser.No. 11/238,574 filed on Sep. 29, 2005, now abandoned, which is acontinuation of U.S. patent application Ser. No. 09/970,543 filed onOct. 4, 2001, now U.S. Pat. No. 7,011,828, which is acontinuation-in-part of U.S. patent application Ser. No. 09/808,382filed on Mar. 14, 2001, now U.S. Pat. No. 7,504,257, which claims thebenefit of priority from Australia Patent Application Nos. PR2920 filedon Feb. 6, 2001, PR1279 filed on Nov. 6, 2000 and PQ6211 filed on Mar.14, 2000. The contents of all of the above applications are incorporatedby reference as if fully set forth herein.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 56379SequenceListing.txt, created on May 1,2013, comprising 6,618 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to undifferentiated human embryonic stemcells, methods of cultivation and propagation and production ofdifferentiated cells. In particular it relates to the production ofhuman ES cells capable of yielding somatic differentiated cells invitro, as well as committed progenitor cells such as neural progenitorcells capable of giving rise to mature somatic cells including neuralcells and/or glial cells and uses thereof.

INTRODUCTION

The production of human embryonic stem cells which can be eithermaintained in an undifferentiated state or directed to undergodifferentiation into extraembryonic or somatic lineages in vitro allowsfor the study of the cellular and molecular biology of early humandevelopment, functional genomics, generation of differentiated cellsfrom the stem cells for use in transplantation or drug screening anddrug discovery in vitro.

In general, stem cells are undifferentiated cells which can give rise toa succession of mature functional cells. For example, a haematopoieticstem cell may give rise to any of the different types of terminallydifferentiated blood cells. Embryonic stem (ES) cells are derived fromthe embryo and are pluripotent, thus possessing the capability ofdeveloping into any organ, cell type or tissue type or, at leastpotentially, into a complete embryo.

The development of mouse ES cells in 1981 (Evans and Kaufman, 1981;Martin, 1981) provided the paradigm, and, much of the technology, forthe development of human ES cells. Development of ES cells evolved outof work on mouse teratocarcinomas, (tumours arising in the gonads of afew inbred strains), which consist of a remarkable array of somatictissues juxtaposed together in a disorganised fashion. Classical work onteratocarcinomas established their origins from germ cells in mice, andprovided the concept of a stem cell (the embryonal carcinoma or EC cell)which could give rise to the multiple types of tissue found in thetumours (Kleinsmith and Pierce, 1964; review, Stevens, 1983). The fieldof teratocarcinoma research (review, Martin, 1980) expanded considerablyin the 70's, as the remarkable developmental capacity of the EC stemcell became apparent following the generation of chimaeric mice byblastocyst injection of EC cells, and investigators began to realise thepotential value of cultured cell lines from the tumours as models formammalian development. EC cells however had limitations. They oftencontained chromosomal abnormalities, and their ability to differentiateinto multiple tissue types was often limited.

Since teratocarcinomas could also be induced by grafting blastocysts toectopic sites, it was reasoned that it might be possible to derivepluripotential cell lines directly from blastocysts rather than fromtumours, as performed in 1981 by Gail Martin and Martin Evansindependently. The result was a stable diploid cell line which couldgenerate every tissue of the adult body, including germ cells.Teratocarcinomas also develop spontaneously from primordial germ cellsin some mouse strains, or following transplantation of primordial germcells to ectopic sites, and in 1992 Brigid Hogan and her colleaguesreported the direct derivation of EG cells from mouse primordial germcells (Matsui et al., 1992). These EG cells have a developmentalcapacity very similar to ES cells.

Testicular teratocarcinomas occur spontaneously in humans, andpluripotential cell lines were also developed from these (review,Andrews, 1988). Two groups reported the derivation of cloned cell linesfrom human teratocarcinoma which could differentiate in vitro intoneurons and other cell types (Andrews et al., 1984, Thompson et al.,1984). Subsequently, cell lines were developed which could differentiateinto tissues representative of all three embryonic germ layers (Pera etal., 1989). As analysis of the properties of human EC cells proceeded,it became clear that they were always aneuploid, usually (though notalways) quite limited in their capacity for spontaneous differentiationinto somatic tissue, and different in phenotype from mouse ES or ECcells.

The properties of the pluripotent cell lines developed by Pera et al.(1989) are as follows:

-   -   Express SSEA-3, SSEA-4, TRA 1-60, GCTM-2, alkaline phosphatase,        Oct-4    -   Grow as flat colonies with distinct cell borders    -   Differentiate into derivatives of all three embryonic germ        layers    -   Feeder cell dependent (feeder cell effect on growth not        reconstituted by conditioned medium from feeder cells or by        feeder cell extracellular matrix)    -   Highly sensitive to dissociation to single cells, poor cloning        efficiency even on a feeder cell layer    -   Do not respond to Leukemia Inhibitory Factor

These studies of human EC cells essentially defined the phenotype ofprimate pluripotential stem cells.

Derivation of primate ES cells from the rhesus monkey blastocyst andlater from that of the marmoset (Thomson et al., 1995, 1996) has beendescribed. These primate cell lines were diploid, but otherwise theyclosely resembled their nearest counterpart, the human EC cell. Theimplication of the monkey work and the work on human EC cells was that apluripotent stem cell, which would be rather different in phenotype froma mouse ES cell, could likely be derived from a human blastocyst.

Bongso and coworkers (1994) reported the short term culture andmaintenance of cells from human embryos fertilised in vitro. The cellsisolated by Bongso and coworkers had the morphology expected ofpluripotent stem cells, but these early studies did not employ feedercell support, and it was impossible to achieve long term maintenance ofthe cultures.

James Thomson and coworkers (1998) derived ES cells from surplusblastocysts donated by couples undergoing treatment for infertility. Themethodology used was not very different from that used 17 years earlierto derive mouse ES stem cells. The trophectoderm, thought to beinhibitory to ES cell establishment, was removed by immunosurgery, theinner cell mass was plated on to a mouse embryonic fibroblast feedercell layer, and following a brief period of attachment and expansion,the resulting outgrowth was disaggregated and replated onto anotherfeeder cell layer. There were no significant departures from mouse ESprotocols in the media or other aspects of the culture system and arelatively high success rate was achieved. The phenotype of the cellswas similar to that outlined above in the human EC studies of Pera etal.

In the studies of Thomson et al. on monkey and human ES cells, there wasno evidence that the cells showed the capacity for somaticdifferentiation in vitro. Evidence for in vitro differentiation waslimited to expression of markers characteristic of trophoblast andendoderm formation (production of human chorionic gonadotrophin andalphafetoprotein). It is not possible to state whether the cells foundproducing alphafetoprotein represent extraembryonic (yolk sac) endodermor definitive (embryonic) endoderm though the former is far more likely.Thus an essential feature for any human ES cell line to be of practicaluse, namely the production of differentiated somatic cells in vitro asseen in previous studies of human EC cells, was not demonstrated in themonkey or human ES cell studies.

Much attention recently has been devoted to the potential applicationsof stem cells in biology and medicine, the properties ofpluripotentiality and immortality are unique to ES cells and enableinvestigators to approach many issues in human biology and medicine forthe first time. ES cells potentially can address the shortage of donortissue for use in transplantation procedures, particularly where noalternative culture system can support growth of the required committedstem cell. However, it must be noted that almost all of the wide rangingpotential applications of ES cell technology in human medicine-basicembryological research, functional genomics, growth factor and drugdiscovery, toxicology, and cell transplantation are based on theassumption that it will be possible to grow ES cells on a large scale,to introduce genetic modifications into them, and to direct theirdifferentiation. Present systems fall short of these goals, but thereare indications of progress to come. The identification of novel factorsdriving pluripotential stem cell growth or stem cell selection protocolsto eliminate the inhibitory influence of differentiated cells, bothoffer a way forward for expansion and cloning of human ES cells.

The mammalian nervous system is a derivative of the ectodermal germlayer of the postimplantation embryo. During the process of axisformation, it is thought that inductive signals elaborated by severalregions of the embryo (the anterior visceral endoderm and the earlygastrula organiser) induce the pluripotent cells of the epiblast toassume an anterior neural fate (Beddington and Robertson, 1999). Themolecular identity of the factors elaborated by these tissues whichdirect neurogenesis is unknown, but there is strong evidence from lowervertebrates that antagonists of the Wnt and BMP families of signallingmolecules may be involved.

Embryonic stem cells are pluripotent cells which are thought tocorrespond to the epiblast of the periimplantation embryo. Mouse EScells are able to give rise to neural tissue in vitro eitherspontaneously or during embryoid body formation. The neural tissue oftenforms in these circumstances in amongst a mixture of a range of celltypes. Alteration of the conditions of culture, or subsequent selectionof neural cells from this mixture, has been used to produce relativelypure populations of neuronal cells from differentiating cultures of EScells (eg Li et al., 1998). These neuronal cells have been used inexperimental models to correct various deficits in animal model systems(review, Svendsen and Smith, 1999). The same has not yet been achievedwith human ES cell derived neurons, though neuronal cells have beenderived from human embryonal carcinoma cells which were induced todifferentiate using retinoic acid. These EC cells were subsequentlyshown to correct deficits in experimental models of CNS disease.

A suitable source of human ES derived neurons would be desirable sincetheir availability would provide real advantages for basic and appliedstudies of CNS development and disease. Controlled differentiation ofhuman ES cells into the neural lineage will allow experimentaldissection of the events during early development of the nervous system,and the identification of new genes and polypeptide factors which mayhave a therapeutic potential such as induction of regenerativeprocesses. Additional pharmaceutical applications may include thecreation of new assays for toxicology and drug discovery, such ashigh-throughput screens for neuroprotective compounds. Generation ofneural progenitors from ES cells in vitro may serve as an unlimitedsource of cells for tissue reconstruction and for the delivery andexpression of genes in the nervous system.

It is an object of the invention to overcome or at least alleviate someof the problems of the prior art.

SUMMARY OF THE INVENTION

In one aspect of the present invention, there is provided an enrichedpreparation of undifferentiated human embryonic stem cells capable ofproliferation in vitro and differentiation to neural progenitor cells,neuron cells and/or glial cells.

Preferably the undifferentiated ES cells have the potential todifferentiate into neural progenitor cells, neuron cells and/or glialcells when subjected to differentiating conditions.

More preferably, the undifferentiated ES cells are capable ofmaintaining an undifferentiated state when cultured on a fibroblastfeeder layer.

In another aspect of the present invention there is provided anundifferentiated human embryonic stem cell wherein the cell isimmunoreactive with markers for human pluripotent stem cells includingSSEA-4, GCTM-2 antigen, TRA 1-60 and wherein said cell may differentiateunder differentiating conditions to neural cells. Preferably, the cellsexpress the transcription factor Oct-4 as demonstrated by RT-PCR. Morepreferably, the cells maintain a diploid karyotype during prolongedcultivation in vitro.

In another aspect there is provided an undifferentiated cell linecapable of differentiation into neural progenitor cells, neurone cellsand glial cells and preferably produced by a method of the presentinvention.

In another aspect there is provided a differentiated committedprogenitor cell line that may be cultivated for prolonged periods andgive rise to large quantities of progenitor cells.

In another aspect there is provided a differentiated committedprogenitor cell line capable of differentiation into mature neuronsand/or glial cells.

In another aspect, there is provided a neural progenitor cell, neuroncell and/or a glial cell differentiated in vitro from anundifferentiated embryonic stem cell. There is also provided a committedneural progenitor cell capable of giving rise to mature neuron cells andglial cells.

In another aspect there is provided a differentiated committedprogenitor cell line capable of establishing a graft in a recipientbrain, to participate in histogenesis of the nervous system and toconstitute the neuronal, astrocyte and oligodendrocyte lineages in vivo.

Preferably, the undifferentiated cell line is preserved by preservationmethods such as cryopreservation. Preferably the method ofcryopreservation is a method highly efficient for use with embryos suchas vitrification. Most preferably, the method includes the Open PulledStraw (OPS) vitrification method.

In another aspect the neural progenitor cell line is preserved bypreservation methods such as cryopreservation.

In another aspect, there is provided a neural progenitor cell capable ofdifferentiating into glial cells, including astrocytes andoligodendrocytes.

In another aspect, there is provided a neural progenitor cell capable oftransdifferentiation into other cell lineages, to generate stem cellsand differentiated cells of non-neuronal phenotype, such ashemangioblast, hematopoietic stem cells, endothelial stem cells,embryonic endoderm and ectodermal cells.

In a further aspect of the present invention, there is provided a methodof preparing undifferentiated human embryonic stem cells fordifferentiation into neural progenitor cells, said method including:

obtaining an in vitro fertilised human embryo and growing the embryo toa blastocyst stage of development;

removing inner cells mass (ICM) cells from the embryo;

culturing ICM cells under conditions which do not induce extraembryonicdifferentiation and cell death and promote proliferation ofundifferentiated cells; and

recovering the stem cells.

In a further preferred embodiment of the present invention there isprovided a method of preparing undifferentiated human embryonic stemcells for differentiation into neural progenitor cells, said methodincluding:

obtaining an in vitro fertilised human embryo;

removing inner cell mass (ICM) cells from the embryo;

culturing ICM cells on a fibroblast feeder layer to promoteproliferation of embryonic stem cells; and

recovering stem cells from the feeder layer.

In a further embodiment of the invention, the method further includes:

replacing the stem cells from the fibroblast feeder layer onto anotherfibroblast feeder layer; and

culturing the stem cells for a period sufficient to promoteproliferation of morphologically undifferentiated stem cells.

In another aspect of the invention the method further includespropagating the undifferentiated stem cells.

In another aspect of the invention there is provided a method ofinducing somatic differentiation of stem cells in vitro into progenitorcells said method comprising:

obtaining undifferentiated stem cells; and

providing a differentiating signal under conditions which arenon-permissive for stem cell renewal, do not kill cells and inducesunidirectional differentiation toward extraembryonic lineages.

In a preferred embodiment of the present invention, there is provided amethod of inducing somatic differentiation of stem cells in vitro intoprogenitor cells, said method comprising:

obtaining undifferentiated stem cells; and

culturing said cells for a prolonged period and at high density on afibroblast feeder cell layer to induce differentiation.

In another preferred embodiment of the present invention, there isprovided a method of inducing somatic differentiation of stem cells invitro into progenitor cells, said method comprising:

obtaining undifferentiated stem cells; and

transferring said cells into serum free media to induce differentiation.

In an additional aspect of the invention method may be used fordirecting stem cells to differentiate toward a somatic lineage.Furthermore, the method allows the establishment of a pure preparationof progenitor cells from a desired lineage and facilitate theestablishment of a pure somatic progenitor cell line.

In another preferred embodiment of the present invention, there isprovided a method of inducing the differentiation of ES derived neuralprogenitor cells into differentiated mature neuronal cells, and glialcells including oligodendrocyte and astrocyte cells.

This invention provides a method that generates an in vitro and in vivomodel of controlled differentiation of ES cells towards the neurallineage. The model, and the cells that are generated along the pathwayof neural differentiation may be used for the study of the cellular andmolecular biology of human neural development, for the discovery ofgenes, growth factors, and differentiation factors that play a role inneural differentiation and regeneration, for drug discovery and for thedevelopment of screening assays for teratogenic, toxic andneuroprotective effects.

In a further aspect of the invention there is provided a neuralprogenitor cell, a neuronal cell and a glial cell that may be used forcell therapy and gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1F show phase contrast micrographs of ES cells and theirdifferentiated progeny. A, inner cell mass three days after plating. B,colony of ES cells. C, higher magnification of an area of an ES cellcolony. D, an area of an ES cell colony undergoing spontaneousdifferentiation during routine passage. E, a colony four days afterplating in the absence of a feeder cell layer but in the presence of2000 units/ml human LIF undergoing differentiation in its periphery. F,neuronal cells in a high density culture. Scale bars: A and C, 25microns; B and E, 100 microns; D and F, 50 microns.

FIGS. 2A-2H show marker expression in ES cells and their differentiatedsomatic progeny. A, ES cell colony showing histochemical staining foralkaline phosphatase. B. ES cell colony stained with antibody MC-813-70recognising the SSEA-4 epitope. C, ES cell colony stained with antibodyTRA1-60. D, ES cell colony stained with antibody GCTM-2. E, high densityculture, cell body and processes of a cell stained withanti-neurofilament 68 kDa protein. F, high density culture, cluster ofcells and network of processes emanating from them stained with antibodyagainst neural cell adhesion molecule. G, high density culture, cellsshowing cytoplasmic filaments stained with antibody to muscle actin. H,high density culture, cell showing cytoplasmic filaments stained withantibody to desmin. Scale bars: A, 100 microns; B-D, and F, 200 microns;E, G and H, 50 microns.

FIGS. 3A-3D show RT-PCR analysis of gene expression in ES cells andtheir differentiated derivatives. All panels show 1.5% agarose gelsstained with ethidium bromide. A, expression of Oct-4 and b-actin in ESstem cells and high density cultures. Lane 1, 100 bpDNA ladder. Lane 2,stem cell culture, b-actin. Lane 3, stem cell culture, Oct-4. Lane 4,stem cell culture, PCR for Oct-4 carried out with omission of reversetranscriptase. Lane 5, high density culture, b-actin. Lane 6, highdensity culture, Oct-4. Lane 7, high density culture, PCR for Oct-4carried out with omission of reverse transcriptase. b-actin band is 200bp and Oct-4 band is 320 bp. B, expression of nestin and Pax-6 in neuralprogenitor cells that were derived from differentiating ES colonies.Left lane, 100 bp DNA ladder; lane 1, b-actin in HX 142 neuroblastomacell line (positive control for nestin PCR); lane 2, b-actin in neuralprogenitor cells; lane 3, nestin in HX 142 neuroblastoma cell line; lane4, nestin in neural progenitor cells; lane 5, nestin PCR on same sampleas lane 4 without addition of reverse transcriptase; lane 6, Pax-6; lane7, Pax-6 PCR on same sample as line 6 without addition of reversetranscriptase. Nestin band is 208 bp, Pax-6 is 274 bp. C, expression ofglutamic acid decarboxylase in cultures of neurons. Left lane, 100 bpDNA ladder; lane 1, b-actin; lane 2, b-actin PCR on same sample as lane1 without addition of reverse transcriptase; lane 3, glutamic aciddecarboxylase; lane 4 glutamic acid decarboxylase on same sample as lane3 without addition of reverse transcriptase. Glutamic acid decarboxylaseband is 284 bp. D, expression of GABA Aα2 receptor. Left lane, 100 bpDNA ladder; lane 1, b-actin; lane 2, GABA Aα2 receptor; lane 3, PCRwithout addition of reverse transcriptase. GABA Aα2 receptor subunitband is 471 bp.

FIGS. 4A-4F show histology of differentiated elements found in teratomasformed in the testis of SCID mice following inoculation of HES-1 orHES-2 colonies. A, cartilage and squamous epithelium, HES-2. B, neuralrosettes, HES-2. C, ganglion, gland and striated muscle, HES-1. D, boneand cartilage, HES-1. E, glandular epithelium, HES-1. F, ciliatedcolumnar epithelium, HES-1. Scale bars: A-E, 100 microns; F, 50 microns.

FIGS. 5A-5D show phase contrast microscopy and immunochemical analysisof marker expression in neural progenitor cells isolated fromdifferentiating ES cultures. A, phase contrast image of a sphere formedin serum-free medium. B-D, indirect immunofluorescence staining ofspheres, 4 hours after plating on adhesive substrate, for N-CAM, nestin,and vimentin respectively. In C and D, cells at the base of the spherewere placed in plane of focus to illustrate filamentous staining;confocal examination revealed that cells throughout the sphere weredecorated by both antibodies. Scale bar is 100 microns in all panels.

FIGS. 6A-6H show phase contrast appearance and marker expression incultures of neurons derived from progenitor cells shown in FIG. 5. A,phase contrast micrograph of differentiated cells emanating from asphere plated onto adhesive surface. B-H, indirect immunofluorescencemicroscopy of differentiated cells decorated with antibodies against 200kDa neurofilament protein (B), 160 kDa neurofilament protein (C),MAP2a+b (D), glutamate (E), synaptophysin (F), glutamic aciddecarboxylase (G) and β-tubulin (H). Scale bars: A; B, 100 microns; C,200 mircons; D, 20 microns; E and F, 10 microns; G, 20 microns; H, 25microns.

FIG. 7 shows neural precursors proliferating as a monolayer on a plastictissue culture dish in the presence of EGF and bFGF. These monolayercultures of proliferating cells were obtained after prolongedcultivation (2-3 weeks) of the spheres in the presence of growth factorswithout sub-culturing.

FIG. 8 shows phase contrast appearance of a culture consisting ofdifferentiated neural cells.

FIG. 9 shows phase contrast appearance of a sphere that is formed 72hours after the transfer of a clump of undifferentiated ES cells intoserum free medium (Scale bar 100 microns).

FIG. 10 shows linear correlation between the volume of spheres and thenumber of progenitor cells within a sphere. Spheres of various diametersthat were generated from differentiating ES colonies and were propagatedfor 14-15 weeks were dissaggregated into single cell suspension and thenumber of cells per sphere was counted.

FIG. 11 shows indirect immunofluorescence staining of a sphere, 4 hoursafter plating on adhesive substrate, for N-CAM. The sphere was generatedby direct transfer of undifferentiated ES cells into serum free mediumand propagation of the resulting spheres for 5 passages. (Scale bar 100microns).

FIG. 12 shows indirect immunofluorescence membraneous staining for N-CAMof single cells at the periphery of a sphere 4 hours after plating onadhesive substrate. The sphere was generated by direct transfer ofundifferentiated ES cells into serum free medium and propagation of theresulting spheres for 5 passages. (Scale bar 25 microns).

FIG. 13 shows indirect immunofluorescence staining of a spheres 4 hoursafter plating on adhesive substrate for the intermediate filamentnestin. Cells at the base of the sphere were placed in plane of focus toillustrate filamentous staining. The sphere was generated by directtransfer of undifferentiated ES cells into serum free medium andpropagation of resulting spheres for 5 passages. (Scale bar 25 microns).

FIG. 14 shows indirect immunofluorescence microscopy of a differentiatedcell decorated with antibodies against the oligodendrocyte progenitormarker O4. (Scale bar 12.5 microns).

FIG. 15 shows indirect immunofluorescence staining of a sphere 4 hoursafter plating on adhesive substrate for the intermediate filamentvimentin. Cells at the base of the sphere were placed in plane of focusto illustrate filamentous staining. The sphere was generated by directtransfer of undifferentiated ES cells into serum free medium andpropagation of resulting spheres for 7 passages. (Scale bar 25 microns).

FIG. 16 shows the growth pattern of spheres that were generated directlyfrom undifferentiated ES cells. Each bar represents the mean (±SD)increment in volume per week of 24 spheres at first to sixteen weeksafter derivation. A more excessive growth rate is evident during thefirst 5 weeks.

FIG. 17 shows persistent growth in the volume of spheres along time.Each bar represents the mean (±SD) increment in volume per week of 24spheres at nine to twenty one weeks after derivation. The spheres weregenerated from differentiating ES colonies.

FIG. 18 shows linear correlation between the volume of spheres and thenumber of progenitor cells within a sphere. Spheres of variousdiameters, that were generated directly from undifferentiated ES cellsand were propagated 5-7 weeks, were dissaggregated into single cellsuspension and the number of cells per sphere was counted.

FIG. 19 shows RT-PCR analysis of gene expression in ES cells (a weekafter passage) and neural spheres derived from differentiating coloniesand directly from undifferentiated ES cell. All panels show 2% agarosegels stained with ethidium bromide. Lanes 1, 2 and 3, Oct-4 in ES cellculture, neural spheres derived from differentiating colonies, neuralspheres derived from undifferentiated ES cells. Lane 4, stem cellculture, PCR for Oct-4 carried out with omission of reversetranscriptase. Lanes 5, 6, and 7, nestin in ES cell culture, neuralspheres derived from differentiating colonies, neural spheres derivedfrom undifferentiated ES cells. Lane 8, stem cell culture, PCR fornestin carried out with omission of reverse transcriptase. Lanes 9, 10and 11, Pax-6 in ES cell culture, neural spheres derived fromdifferentiating colonies, neural spheres derived from undifferentiatedES cells. Lane 12, stem cell culture, PCR for Pax-6 carried out withomission of reverse transcriptase. Lane 13, 100 bp DNA ladder. Oct-4band is 320 bp, nestin is 208 bp and Pax-6 is 274 bp.

FIG. 20 shows indirect immunofluorescence microscopy of differentiatedastrocyte cells decorated with antibody against GFAP. (Scale bar 25microns).

FIGS. 21A-21B show indirect immunofluorescence microscopy of brainsections of two mice (A and B) 4 weeks after transplantation of humanneural precursors prelabeled with BrDU. Cells with a nucleus decoratedwith anti BrDU (brown stain, black arrow) are evident near theventricular surface (white arrow indicate mouse unstained nuclei, bar=20microns).

FIGS. 22A-22C show indirect immunofluorescence microscopy of brainsections of a mice 4 weeks after transplantation of human neuralprecursors prelabeled with BrDU. Wide spread distribution oftransplanted human cells decorated by anti BrDU antibodies is evident inthe periventricular areas. The periventricular area in A is demonstratedat a higher magnification in B and C. (Bars=150, 60 and 30 microns in A,B and C).

FIG. 23 shows indirect immunocytochemical microscopy of brain sectionsof a mice 4 weeks after transplantation of human neural precursorsprelabeled with BrDU. The transplanted human cells are migrating alongthe rostral migratory stream (bar=150 microns).

FIGS. 24A-24B show RT-PCR analysis of gene expression in neural spheresderived from differentiating (A) and undifferentiated (B) ES cells. Allpanels show 2% agarose gels stained with ethidium bromide. Lanes 1 and10, 100 bpDNA ladder; Lane 2, CD-34; Lane 3, Flk-1; lane 4, HNF-3; lane5, alfafetoprotein. Lanes 6-9 PCR reaction on the same samples as lanes2-5 carried out with the omission of reverse transcriptase. CD-34 bandis 200 bp, Flk-1 is 199, HNF-3 is 390, AFP is 340 bp.

FIG. 25 shows by RT-PCR analysis the expression of GFAP and the plp genein differentiated cells from neural spheres derived from differentiatingES cell colonies. The expression of GFAP indicates differentiation intoastrocytes while the presence of both dm-20 and plp transcripts indicatethat differentiation into oligodendrocyte cells has occurred. Lanes 2,4, 6 and lanes 3, 5, 7 are from two separate RNA samples fromdifferentiated spheres that were independently derived from ES cells.Lane 1 and 8, 100 bp DNA ladder; Lanes 2 and 4, GFAP; lanes 3 and 5, plpand dm-20; lanes 6 and 7, PCR reaction on the same samples as lanes 3and 5 carried out with the omission of reverse transcriptase. GFAP bandis 383, plp band is 354 bp and dm-20 is 249 bp.

FIG. 26 shows a dark field stereomicroscopic photograph of areas(arrows) destined to give rise to neural precursors in a differentiatingES cell colony 3 weeks after passage (bar=1.6 mm).

FIGS. 27A-27C show indirect immunochemical analysis of marker expressionin cultures of neurons derived from progenitor cells that were deriveddirectly from undifferentiated ES cells: A, indirect immunofluorescencemicroscopy of neurits decorated with antibody against 160 kDaneurofilament protein. B and C, indirect immunofluorescence staining ofdifferentiated cells for MAP2a+b and β-tubulin III. Scale bars: A 100microns, B and C 10 microns.

FIGS. 28A-28B show indirect immunochemical analysis of the expression oftyrosine hydroxylase. Neurits (A) and a differentiated cell (B) aredecorated with antibodies against tyrosine hydroxylase. Scale bars: 30microns.

FIGS. 29A-29B show in vivo differentiation into astrocyte cells oftransplanted human neural progenitors prelabeled with BrDU. Donor cellsare identified by indirect immunochemical detection of BrDU (darknuclei, arrows). Duel staining demonstrates donor cells decorated byanti GFAP (orange). Transplanted cells are migrating into the brainparenchyma (white arrow) and are also found in the periventricular zone(dark arrow) (A), A higher magnification of cells that havedifferentiated into astrocytes and migrated into the host brain (B).

FIG. 30 shows in vivo differentiation into oligodendrocyte cells oftransplanted human neural progenitors prelabeled with BrDU. Donor cellsare identified by indirect immunochemical detection of BrDU (darknuclei, arrows). Duel staining demonstrates donor cells decorated byanti CNPase (orange).

FIG. 31 shows cumulative growth curve for human neural progenitorsderived from differentiating colonies. (A) Continuous growth is evidentduring an 18-22 week period. The increment in the volume of the sphereswas continuously monitored as an indirect measure of the increase incell numbers. A linear positive correlation between the volume of thespheres and the number of cells within the spheres (B, insert) wasmaintained along cultivation. It supported the validity of monitoringthe increment of sphere volume as an indirect indicator of cellproliferation.

FIG. 32 shows RT-PCR analysis of the expression of non-neural markers inhuman ES derived spheres. All panels show 2% agarose gels stained withethidium bromide. The symbols + and − indicate whether the PCR reactionwas performed with or without the addition of reverse transcriptase. A 1Kb plus DNA ladder was used in all panels. β-actin band is 291 bp,keratin is 780 bp, Flk-1 is 199 bp, CD34 is 200 bp, AC-133 is 200 bp,transferin is 367 bp, amylase is 490 bp and α1 anti trypsin is 360 bp.

FIG. 33 shows a phase contrast micrograph of differentiated cellsgrowing out from a sphere 2 weeks after plating onto an adhesive surfaceand culture in the absence of growth factors. Scale bar is 200 μm.

FIG. 34 shows RT-PCR analysis of the expression of neuronal and glialmarkers in differentiated cells originating from human ES derived neuralspheres. All panels show 2% agarose gels stained with ethidium bromide.The symbols + and − indicate whether the PCR reaction was performed withor without the addition of reverse transcriptase. A 1 Kb plus DNA ladderwas used in all panels. Plp and dm-20 bands are 354 bp and 249 bprespectively, MBP is 379 bp, GFAP is 383 bp, NSE is 254 bp and NF-M is430 bp.

FIGS. 35A-35B show indirect immunochemical analysis of the expression ofserotonin (A) and GABA (B). Scale bars are 20 μm.

FIGS. 36A-36E show dissemination of transplanted BrdU+ human ES-derivedneural progenitor cells in the mouse host brain.

(A) At 2 days after transplantation most cells were found lining theventricular wall. (B) After 4-6 weeks most cells had left the ventricles(V) and populated the corpus callosum (CC), fimbria (fim), internalcapsule (i.c.). BrdU+ cells were not found in the striatum (str) or CAregion of the hippocampus (hipp). (C) Chains of BrdU+ cells were foundin the rostral migratory stream (RMS). (D) BrdU+ cells in theperiventricular white matter. (E) Higher magnification of D, to shownuclear specific localization of BrdU; and

FIGS. 37A-37G show identification of the transplanted cells in the brainby human and neural-lineage specific markers. (A) A typical chain oftransplanted cells in the corpus callosum, stained with human specificanti-mitochondrial antibody. The mitochondrial staining (greenfluorescence) on Nomarsky background (blue, cell nuclei indicated byasterisk) shows a typical perinuclear localization. (B) Double stainingfor BrdU (green fluorescence) and human specific anti ribonuclearprotein (red fluorescence) shows nuclear co-localization, indicatingthat BrdU+ cells were indeed of human origin. (C) A GFAP+ astrocyte(red) from the periventricular region, co-labeled with BrdU (green),indicating its origin from the graft. (D) An NG2+ oligodendrocyteprogenitor (red) in the periventricular region, co-labeled with BrdU(green). (E) A CNPase+ oligodendrocyte (red) in the corpus callosum,co-labeled with BrdU (immunohistochemistry, shown as dark nucleus inNomarsky). (F) Neuronal processes in the fimbria, stained with a humanspecific anti-70 kDa neurofilament. (G) A βIII-tubulin+ neuron (greenfluorescence) in the olfactory bulb, co-labeled with BrdU (as darknucleus (arrow) in Nomarsky). Bars=10 μm.

DESCRIPTION OF THE INVENTION

In one aspect of the present invention there is provided an enrichedpreparation of human undifferentiated embryonic stem cells capable ofproliferation in vitro and differentiation to neural progenitor cells,neuron cells and/or glial cells.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises”, is not intended to exclude other additives, components,integers or steps.

Established pluripotent ES cell lines from human blastocysts are shownin, PCT/AU99/00990 by the applicants. In contrast to data that haspublished previously, the human ES cell lines that have been derived bythe applicants in the present application have been shown todifferentiate in vitro into somatic lineages and give rise to neuronsand muscle cells. Moreover, Applicants have demonstrated the derivationof neural progenitor cells from human ES cells in vitro. These ESderived human neural progenitors may give rise to mature neurons invitro. The contents of PCT/AU99/00990 are hereby incorporated.

Proliferation in vitro may include cultivation of the cells forprolonged periods. The cells are substantially maintained in anundifferentiated state. Preferably the cells are maintained underconditions which do not induce cell death or extraembryonicdifferentiation.

Preferably, they are capable of maintaining an undifferentiated statewhen cultured on a fibroblast feeder layer preferably undernon-differentiating conditions. Preferably the fibroblast feeder layerdoes not induce extraembryonic differentiation.

More preferably the cells have the potential to differentiate in vitrowhen subjected to differentiating conditions. Most preferably the cellshave the capacity to differentiate in vitro into a wide array of somaticlineages.

The promotion of stem cells capable of being maintained in anundifferentiated state in vitro on one hand, and which are capable ofdifferentiation in vitro into extraembryonic and somatic lineages on theother hand, allows for the study of the cellular and molecular biologyof early human development, functional genomics, generation ofdifferentiated cells from the stem cells for use in transplantation ordrug screening and drug discovery in vitro.

Once the cells are maintained in the undifferentiated state, they may bedifferentiated to mature functional cells. The embryonic stem cells arederived from the embryo and are pluripotent and have the capability ofdeveloping into any organ or tissue type. Preferably the tissue type isselected from the group including endocrine cells, blood cells, neuralcells or muscle cells. Most preferably they are neural cells.

In another aspect of the present invention there is provided anundifferentiated human embryonic stem cell wherein the cell isimmunoreactive with markers for human pluripotent stem cells includingSSEA-4, GCTM-2 antigen, and TRA 1-60 and wherein said cell candifferentiate, under differentiating conditions to neural cells.Preferably, the cells express specific transcription factors such asOct-4 as demonstrated by RT-PCR, or methods of analysis of differentialgene expression, microarray analysis or related techniques. Morepreferably the cells maintain a diploid karyotype during prolongedcultivation in vitro. Preferably, the stem cell will constitute anenriched preparation of an undifferentiated stem cell line. Morepreferably, the stem cell line is a permanent cell line, distinguishedby the characteristics identified above. They preferably have normalkaryotype along with the characteristics identified above. Thiscombination of defining properties will identify the cell lines of theinvention regardless of the method used for their isolation.

Methods of identifying these characteristics may be by any method knownto the skilled addressee. Methods such as (but not limited to) indirectimmunoflourescence or immunocytochemical staining may be carried out oncolonies of ES cells which are fixed by conventional fixation protocolsthen stained using antibodies against stem cell specific antibodies andvisualised using secondary antibodies conjugated to fluorescent dyes orenzymes which can produce insoluble colored products. Alternatively, RNAmay be isolated from the stem cells and RT-PCR or Northern blot analysiscarried out to determine expression of stem cell specific genes such asOct-4.

In a preferred embodiment the undifferentiated cells form tumours wheninjected in the testis of immunodeprived SCID mice. These tumoursinclude differentiated cells representative of all three germ layers.The germ layers are preferably endoderm, mesoderm and ectoderm.Preferably, once the tumours are established, they may be disassociatedand specific differentiated cell types may be identified or selected byany methods available to the skilled addressee. For instance, lineagespecific markers may be used through the use of fluorescent activatedcell sorting (FACS) or other sorting method or by direct microdissection of tissues of interest. These differentiated cells may beused in any manner. They may be cultivated in vitro to produce largenumbers of differentiated cells that could be used for transplantationor for use in drug screening for example.

In another aspect there is provided a differentiated committedprogenitor cell line capable of differentiation and propagation intomature neurons and/or glial cells. The undifferentiated cells maydifferentiate in vitro to form neural progenitor cells, neuron cellsand/or glial cells.

In another aspect, there is provided a neural progenitor cell, neuroncell and/or glial cell differentiated in vitro from an undifferentiatedembryonic stem cell. There is also provided a committed neuralprogenitor cell capable of giving rise to mature neuron cells. Themature neuron cell may be characterized by an ability to express 160 kDaneuro-filament protein, MAP2ab, glutamate, synaptophysin, glutamic aciddecarboxylase (GAD), tyrosine hydroxylase, GABA and serotonin.

In another aspect, there is provided a neural progenitor cell capable ofdifferentiating into glial cells, including astrocytes andoligodendrocytes. The glial cells include microglial cells and radialglial cells.

In another aspect, there is provided a neural progenitor cell capable oftransdifferentiation into other cell lineages, to generate stem cellsand differentiated cells of non-neuronal phenotype, such ashemangioblast hematopoietic stem cells or endothelial stem cells,embryonic endoderm cells and ectoderm cells.

These cells may be obtained by somatic differentiation of human EScells, identified by neural markers. These cells may be isolated in pureform from differentiating ES cells, in vitro, and propagated in vitro.They may be induced to under go differentiation to mature neurons and/orglial cells.

The cells may undergo differentiation in vitro to yield neuralprogenitor cells, neuron or glial cells as well as extraembryonic cells,such differentiation being characterised by novel gene expressioncharacteristic of specific lineages as demonstrated byimmunocytochemical or RNA analysis. Characterisation may be obtained byusing expression of genes characteristic of pluripotent cells orparticular lineages. Preferably, differential expression of Oct-4 may beused to identify stem cells from differentiated cells. Otherwise, thepresence or absence of expression of other genes characteristic ofpluripotent stem cells or other lineages may include Genesis, GDF-3 orCripto. Analysis of these gene expressions may create a gene expressionprofile to define the molecular phenotype of an ES cell, a committedprogenitor cell, or a mature differentiated cell of any type. Suchanalysis of specific gene expression in defined populations of cellsfrom ES cultures is called cytomics. Methods of analysis of geneexpression profiles include RT-PCR, methods of differential geneexpression, microarray analysis or related techniques.

Differentiating cultures of the stem cells secrete human chorionicgonadotrophin (hCG) and α-fetoprotein (AFP) into culture medium, asdetermined by enzyme-linked immunosorbent assay carried out on culturesupernatants. Hence this may also serve as a means of identifying thedifferentiated cells.

The differentiated cells forming neural progenitor cells, neuron cellsand/or glial cells may also be characterised by expressed markerscharacteristic of differentiating cells. The in vitro differentiatedcell culture may be identified by detecting molecules such as markers ofthe neuroectodermal lineage, markers of neural progenitor cells,neuro-filament proteins, monoclonal antibodies such as MAP2ab,glutamate, synaptophysin, glutamic acid decarboxylase, GABA, serotonin,tyrosine hydroxylase, β-tubulin, β-tubulin III, GABA Aα2 receptor, glialfibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide3′-phosphodiesterase (CNPase), plp, DM-20, O4, and NG-2 staining.

In another preferred aspect of the present invention there is provided aneural progenitor cell wherein the cell express markers for theneuroectodermal lineage as well as neural markers selected from thegroup including polysialyated N-CAM, N-CAM, A2B5, nestin, vimentin andthe transcriptional factor Pax-6, and do not express Oct-4.

Preferably, the cells do not express the transcriptional factor OCT-4.This may be demonstrated by RT-PCR, or methods of analysis ofdifferential gene expression, microarray analysis or related techniques.More preferably the cells will constitute an enriched preparation. Theycan proliferate in vitro for prolonged periods at an undifferentiatedneural progenitor state to produce a large number of cells. The neuralprogenitor cells can differentiate, under differentiating conditions tomature neurons and glial cells.

In yet another aspect, the invention provides a neural progenitor cellwhich is capable of establishing a graft in a recipient brain.Preferably the neural progenitor cell is as described above.

Upon transplantation to the developing brain they incorporateextensively into the host brain, demonstrate wide spread distribution,migrate along established host brain migratory tracks, differentiate ina region specific manner into progeny of the three fundamental neurallineages, indicating their capability to respond to local cues andparticipate in the development and histogenesis of the living host. Thiscombination of defining properties will identify the neural progenitorcell lines of the invention regardless of the method used for theirisolation.

In yet another aspect of the present invention, there is provided aglial cell differentiated from a neural progenitor cell. Preferably, theglial cell is an astrocyte or an oligodendrocyte. Oligodendrocytes maybe identified by O4 and NG-2 immunostaining or by RNA transcripts ofmyelin basic protein (MBP), plp and dm-20.

In a further aspect of the invention, there is provided a method ofpreparing undifferentiated human embryonic stem cells fordifferentiation into neural progenitor cells, said method including:

obtaining an in vitro fertilised human embryo and growing the embryo toa blastocyst stage of development;

removing inner cells mass (ICM) cells from the embryo;

culturing ICM cells under conditions which do not induce extraembryonicdifferentiation and cell death, and promote proliferation ofundifferentiated stem cells; and

recovering the stem cells.

The stem cells will be undifferentiated cells and can be induced todifferentiate when a differentiating signal is applied.

In a preferred embodiment of the present invention there is provided amethod of preparing undifferentiated human embryonic stem cells fordifferentiation into neural progenitor cells, said method including:

obtaining an in vitro fertilised human embryo;

removing inner cells mass (ICM) cells from the embryo;

culturing ICM cells on a fibroblast feeder layer to promoteproliferation of embryonic stem cells; and

recovering stem cells from the feeder layer.

Embryonic stem cells (ES) are derived from the embryo. These cells areundifferentiated and have the capability of differentiation to a varietyof cell types. The “embryo” is defined as any stage after fertilizationup to 8 weeks post conception. It develops from repeated division ofcells and includes the stages of a blastocyst stage which comprises anouter trophectoderm and an inner cell mass (ICM).

The embryo required in the present method may be an in vitro fertilisedembryo or it may be an embryo derived by transfer of a somatic cell orcell nucleus into an enucleated oocyte of human or non human originwhich is then activated and allowed to develop to the blastocyst stage.

The embryo may be fertilised by any in vitro methods available. Forinstance, the embryo may be fertilised by using conventionalinsemination, or intracytoplasmic sperm injection.

An embryo that is recovered from cryopreservation is also suitable. Anembryo that has been cryopreserved at any stage of development issuitable. Preferably embryos that were cryopreserved at the zygote orcleavage stage are used. Any method of cryopreservation of embryos maybe used. It is preferred that a method producing high quality (goodmorphological grade) embryos is employed.

It is preferred that any embryo culture method is employed but it ismost preferred that a method producing high quality (good morphologicalgrade) blastocysts is employed. The high quality of the embryo can beassessed by morphological criteria. Most preferably the inner cell massis well developed. These criteria can be assessed by the skilledaddressee.

Following insemination, embryos may be cultured to the blastocyst stage.Embryo quality at this stage may be assessed to determine suitableembryos for deriving ICM cells. The embryos may be cultured in anymedium that maintains their survival and enhances blastocystdevelopment.

Preferably, the embryos are cultured in droplets under pre-equilibratedsterile mineral oil in IVF-50 or Scandinavian 1 (S1) or G1.2 medium(Scandinavian IVF). Preferably the incubation is for two days. If IVF-50or S1 is used, on the third day, an appropriate medium such as a mixtureof 1:1 of IVF-50 and Scandinavian-2 medium (Scandinavian IVF) may beused. From at least the fourth day, a suitable medium such as G2.2 orScandinavian-2 (S2) medium may be used solely to grow the embryos toblastocyst stage (blastocysts). Preferably, only G2.2 medium is usedfrom the fourth day onwards.

In a preferred embodiment, the blastocyst is subjected to enzymaticdigestion to remove the zona pellucida or a portion thereof. Preferablythe blastocyst is subjected to the digestion at an expanded blastocyststage which may be approximately on day 6. Generally this is atapproximately six days after insemination.

Any protein enzyme may be used to digest the zona pellucida or portionthereof from the blastocyst. Examples include pronase, acid Tyrodessolution, and mechanical methods such as laser dissection.

Preferably, Pronase is used. The pronase may be dissolved in PBS and G2or S2 medium. Preferably the PBS and Scandinavian-2 medium is diluted1:1. For digestion of zona pellucida from the blastocyst, approximately10 units/ml of Pronase may be used for a period sufficient to remove thezona pellucida. Preferably approximately 1 to 2 mins, more preferably 1to 1.5 mins is used.

The embryo (expanded blastocyst) may be washed in G2.2 or S2 medium, andfurther incubated to dissolve the zona pellucida. Preferably, furtherdigestion steps may be used to completely dissolve the zona. Morepreferably the embryos are further incubated in pronase solution for 15seconds. Removal of the zona pellucida thereby exposes thetrophectoderm.

In a preferred embodiment of the invention the method further includesthe following steps to obtain the inner cell mass cell, said stepsincluding:

treating the embryo to dislodge the trophectoderm of the embryo or aportion thereof;

washing the embryo with a G2.2 or S2 medium to dislodge thetrophectoderm or a portion thereof; and

obtaining inner cell mass cells of the embryo.

Having had removed the zona pellucida, the ICM and trophectoderm becomeaccessible. Preferably the trophectoderm is separated from the ICM. Anymethod may be employed to separate the trophectoderm from the ICM.Preferably the embryo (or blastocyst devoid of zona pellucida) issubjected to immuno-surgery. Preferably it is treated with an antibodyor antiserum reactive with epitopes on the surface of the trophectoderm.More preferably, the treatment of the embryo, (preferably an embryo atthe blastocyst stage devoid of zona pellucida) is combined withtreatment with complement. The antibody and/or antiserum and complementtreatment may be used separately or together. Preferred combinations ofantibody and/or antiserum and complement include anti-placental alkalinephosphatase antibody and Baby Rabbit complement (Serotec) or anti-humanserum antibody (Sigma) combined with Guinea Pig complement (Gibco).

Preferably the antibodies and complement are diluted in G2.2 or S2medium. The antibodies and complement, excluding anti-placental alkalinephosphate (anti-AP) are diluted 1:5 whereas anti-AP antibody is diluted1:20 with S-2 medium.

Preferably the embryo or blastocyst (preferably having the zonapellucida removed) is subjected to the antibody before it is subjectedto the complement. Preferably, the embryo or blastocyst is cultured inthe antibody for a period of approximately 30 mins.

Following the antibody exposure, it is preferred that the embryo iswashed. Preferably it is washed in G2.2 or S2 medium. The embryo orblastocyst preferably is then subjected to complement, preferably for aperiod of approximately 30 mins.

G2.2 or S2 (Scandinavian-2) medium is preferably used to wash the embryoor blastocyst to dislodge the trophectoderm or a portion thereof.Dislodgment may be by mechanical means. Preferably the dislodgment is bypipetting the blastocyst through a small bore pipette.

The ICM cells may then be exposed and ready for removal and culturing.Culturing of the ICM cells may be conducted on a fibroblast feederlayer. In the absence of a fibroblast feeder layer, the cells willdifferentiate. Leukaemia inhibitory factor (LIF) has been shown toreplace the feeder layer in some cases and maintain the cells in anundifferentiated state. However, this seems to only work for mousecells. For human cells, high concentrations of LIF were unable tomaintain the cells in an undifferentiated state in the absence of afibroblast feeder layer.

The conditions which do not induce extraembryonic differentiation andcell death may include cultivating the embryonic stem cells on afibroblast feeder layer which does not induce extraembryonicdifferentiation and cell death.

Mouse or human fibroblasts are preferably used. They may be usedseparately or in combination. Human fibroblasts provide support for stemcells, but they create a non-even and sometimes non-stable feeder layer.However, they may combine effectively with mouse fibroblasts to obtainan optimal stem cell growth and inhibition of differentiation.

The cell density of the fibroblast layer affects its stability andperformance. A density of approximately 25,000 human and 70,000 mousecells per cm² is most preferred. Mouse fibroblasts alone are used at75,000-100,000/cm². The feeder layers are preferably established 6-48hours prior to addition of ES or ICM cells.

Preferably the mouse or human fibroblast cells are low passage numbercells. The quality of the fibroblast cells affects their ability tosupport the stem cells. Embryonic fibroblasts are preferred. For mousecells, they may be obtained from 13.5 day old foetuses. Humanfibroblasts may be derived from embryonic or foetal tissue fromtermination of pregnancy and may be cultivated using standard protocolsof cell culture.

The guidelines for handling the mouse embryonic fibroblasts may includeminimising the use of trypsin digestion and avoidance of overcrowding inthe culture. Embryonic fibroblasts that are not handled accordingly willfail to support the growth of undifferentiated ES cells. Each batch ofnewly derived mouse embryonic fibroblasts is tested to confirm itssuitability for support and maintenance of stem cells.

Fresh primary embryonic fibroblasts are preferred in supporting stemcell renewal and/or induction of somatic differentiation as compared tofrozen-thawed fibroblasts. Nevertheless, some batches will retain theirsupportive potential after repeated freezing and thawing. Therefore eachfresh batch that has proved efficient in supporting ES cells renewaland/or induction of somatic differentiation is retested after freezingand thawing. Batches that retain their potential after freezing andthawing are most preferably used. Batches are tested to determinesuitability for the support of stem cell renewal, the induction ofsomatic differentiation or the induction of extraembryonicdifferentiation.

Some mouse strains yield embryonic fibroblasts which are more suitablefor stem cell maintenance and induction of somatic differentiation thanthose of other strains. For example, fibroblasts derived from inbred129/Sv or CBA mice or mice from a cross of 129/Sv with C57/B16 strainshave proven highly suitable for stem cell maintenance.

Isolated ICM masses may be plated and grown in culture conditionssuitable for human stem cells.

It is preferred that the feeder cells are treated to arrest theirgrowth. Several methods are available. It is preferred that they areirradiated or are treated with chemicals such as mitomycin C thatarrests their growth. Most preferably, the fibroblast feeder cells aretreated with mitomycin C (Sigma).

The fibroblast feeder layer may be generally plated on a gelatin treateddish. Preferably, the tissue culture dish is treated with 0.1% gelatin.

The fibroblast feeder layer may also contain modified fibroblasts. Forinstance, fibroblasts expressing recombinant membrane bound factorsessential for stem cell renewal may be used. Such factors may includefor example human multipotent stem cell factor.

Inner cell mass cells may be cultured on the fibroblast feeder layer andmaintained in an ES medium. A suitable medium is DMEM (GIBCO, withoutsodium pyruvate, with glucose 4500 mg/L) supplemented with 20% FBS(Hyclone, Utah), (betamercaptoethanol—0.1 mM (GIBCO), non essentialamino acids—NEAA 1% (GIBCO), glutamine 2 mM. (GIBCO), and penicillin 50μ/ml, streptomycin 50 μg/ml (GIBCO). In the early stages of ES cellcultivation, the medium may be supplemented with human recombinantleukemia inhibitory factor hLIF preferably at 2000 μ/ml. However, LIFgenerally is not necessary. Any medium may be used that can support theES cells.

The ES medium may be further supplemented with soluble growth factorswhich promote stem cell growth or survival or inhibit stem celldifferentiation. Examples of such factors include human multipotent stemcell factor, or embryonic stem cell renewal factor.

The isolated ICM may be cultured for at least six days. At this stage, acolony of cells develops. This colony is comprised principally ofundifferentiated stem cells. They may exist on top of differentiatedcells. Isolation of the undifferentiated cells may be achieved bychemical or mechanical means or both. Preferably mechanical isolationand removal by a micropipette is used. Mechanical isolation may becombined with a chemical or enzymatic treatment to aid with dissociationof the cells, such as Ca²⁺/Mg²⁺ free PBS medium or dispase.

In a further preferred embodiment of the invention, the method furtherincludes:

replating the stem cells from the fibroblast feeder layer onto anotherfibroblast feeder layer; and

culturing the stem cells for a period sufficient to obtain proliferationof morphologically undifferentiated stem cells.

A further replating of the undifferentiated stem cells is performed. Theisolated clumps of cells from the first fibroblast feeder layer may bereplated on fresh human/mouse fibroblast feeder layer in the same mediumas described above.

Preferably, the cells are cultured for a period of 7-14 days. After thisperiod, colonies of undifferentiated stem cells may be observed. Thestem cells may be morphologically identified preferably by the highnuclear/cytoplasmic ratios, prominent nucleoli and compact colonyformation. The cell borders are often distinct and the colonies areoften flatter than mouse ES cells. The colonies resemble those formed bypluripotent human embryonal carcinoma cell lines such as GCT 27 X-1.

In another embodiment of the invention, the method further includespropagating the undifferentiated stem cells. The methods of propagationmay initially involve removing clumps of undifferentiated stem cellsfrom colonies of cells. The dispersion is preferably by chemical ormechanical means or both. More preferably, the cells are washed in aCa²⁺/Mg²⁺ free PBS or they are mechanically severed from the colonies ora combination of these methods or any known methods available to theskilled addressee. In these methods, cells may be propagated as clumpsof about 100 cells about every 7 days.

In the first method, Ca²⁺/Mg²⁺ free PBS medium may be used to reducecell-cell attachments. Following about 15-20 minutes, cells graduallystart to dissociate from the monolayer and from each other and desiredsize clumps can be isolated. When cell dissociation is partial,mechanical dissociation using the sharp edge of the pipette may assistwith cutting and the isolation of the clumps.

An alternative chemical method may include the use of an enzyme. Theenzyme may be used alone or in combination with a mechanical method.Preferably, the enzyme is dispase.

An alternative approach includes the combined use of mechanical cuttingof the colonies followed by isolation of the subcolonies by dispase.Cutting of the colonies may be performed in PBS containing Ca²⁺ andMg²⁺. The sharp edge of a micropipette may be used to cut the coloniesto clumps of about 100 cells. The pipette may be used to scrape andremove areas of the colonies. The PBS is preferably changed to regularequilibrated human stem cell medium containing dispase (Gibco) 10 mg/mland incubated for approximately 5 minutes at 37° C. in a humidifiedatmosphere containing 5% CO₂. As soon as the clumps detached they may bepicked up by a wide bore micro-pipette, washed in PBS containing Ca²⁺and Mg²⁺ and transferred to a fresh fibroblast feeder layer.

The fibroblast feeder layer may be as described above.

Undifferentiated embryonic stem cells have a characteristic morphologyas described above. Other means of identifying the stem cells may be bycell markers or by measuring expression of genes characteristic ofpluripotent cells.

Examples of genes characteristic of pluripotent cells or particularlineages may include (but are not limited to) Oct-4 and Pax-6,polysialyated N-CAM, N-CAM, A2B5, nestin and vimentin as markers of stemcells and neuronal precursors respectively. Other genes characteristicof stem cells may include Genesis, GDF-3 and Cripto. CD-34 ischaracteristic of hematopoietic stem cells and flk-1 is expressed by thehemangioblast. AC-133 may be characteristic of both hematopoietic andneural progenitors. Keratin is characteristic of epidermal cells whiletransferin, amylase and α1 anti-trypsin are characteristic of embryonicendoderm. Such gene expression profiles may be attained by any methodincluding RT-PCR, methods of differential gene expression, microarrayanalysis or related techniques.

Preferably the stem cells may be identified by being immunoreactive withmarkers for human pluripotent stem cells including SSEA-4, GCTM-2antigen, TRA 1-60. Preferably the cells express the transcription factorOct-4. The cells also maintain a diploid karyotype.

Preferably the neural progenitor cells are identified by expressedmarkers of primitive neuroectoderm and neural stem cells such as N-CAM,polysialyated N-CAM, A2B5, intermediate filament proteins such as nestinand vimentin and the transcription factor Pax-6. Neurons may beidentified by structural markers such as β-tubulin, β-tubulin III, the68 kDa and the 200 kDa neurofilament proteins. Mature neurons may alsobe identified by the 160 kDa neurofilament proteins, Map-2a, b andsynaptophysin, glutamate, GABA, serotonin, tyrosine hydroxylase, GABAbiosynthesis and receptor subunits characteristic of GABA minergicneurons (GABA Aα2). Astrocytes may be identified by the expression ofglial fibrillary acidic protein (GFAP), and oligodendrocyte by2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), plp, DM-20,myelin basic protein (MBP), NG-2 staining and O4.

The stem cells may be further modified at any stage of isolation. Theymay be genetically modified through introduction of vectors expressing aselectable marker under the control of a stem cell specific promotersuch as Oct-4. Some differentiated progeny of embryonic stem cells mayproduce products that are inhibitory to stem cell renewal or survival.Therefore selection against such differentiated cells, facilitated bythe introduction of a construct such as that described above, maypromote stem cell growth and prevent differentiation.

The stem cells may be genetically modified at any stage with markers sothat the markers are carried through to any stage of cultivation. Themarkers may be used to purify the differentiated or undifferentiatedstem cell population at any stage of cultivation.

Genetic construct may be inserted to undifferentiated or differentiatedcells at any stage of cultivation. The genetically modified cells may beused after transplantation to carry and express genes in target organsin the course of gene therapy.

Progress of the stem cells and their maintenance in a differentiated orundifferentiated stage may be monitored in a quantitative fashion by themeasurement of stem cell specific secreted products into the culturemedium or in fixed preparations of the cells using ELISA or relatedtechniques. Such stem cell specific products might include the solubleform of the CD30 antigen or the GCTM-2 antigen or they may be monitoredas described above using cell markers or gene expression.

In another aspect of the invention there is provided a method ofinducing somatic differentiation of stem cells in vitro into progenitorcells said method comprising:

obtaining undifferentiated stem cells; and

providing a differentiating signal under conditions which arenon-permissive for stem cell renewal, do not kill cells and/or inducesunidirectional differentiation toward extraembryonic lineages.

The undifferentiated cell lines of the present invention may be culturedindefinitely until a differentiating signal is given. Preferably, theyare cultured under the conditions described above.

In the presence of a differentiation signal, undifferentiated ES cellsin the right conditions will differentiate into derivatives of theembryonic germ layers (endoderm, mesoderm and ectoderm) such as neurontissue, and/or extraembryonic tissues. This differentiation process canbe controlled.

This method is useful for directing stem cells to differentiate toward asomatic lineage. Furthermore, the method allows the establishment of apure preparation of progenitor cells from a desired lineage and theelimination of unwanted differentiated cells from other lineages. Themethod facilitates the establishment of a pure somatic progenitor cellline.

The method may be used to derive an enriched preparation of a variety ofsomatic progenitors such as but not limited to mesodermal progenitors(such as hemangioblast or hematopoietic stem cells) and neuralprogenitors. Preferably the method is used to derive neural progenitors.

Conditions for obtaining differentiated cultures of somatic cells fromembryonic stem cells are those which are non-permissive for stem cellrenewal, but do not kill stem cells or drive them to differentiateexclusively into extraembryonic lineages. A gradual withdrawal fromoptimal conditions for stem cell growth favours somatic differentiation.The stem cells are initially in an undifferentiated state and can beinduced to differentiate.

In a preferred embodiment of the present invention, there is provided amethod of inducing somatic differentiation of stem cells in vitro intoprogenitor cells, said method comprising:

obtaining undifferentiated stem cells; and

culturing said cells for prolonged periods and at high density on afibroblast feeder cell layer to induce differentiation.

In another preferred embodiment of the present invention, there isprovided a method of inducing somatic differentiation of stem cells invitro into progenitor cells, said method comprising:

obtaining undifferentiated stem cells; and

transferring said cells into serum free media to induce differentiation.

The stem cells may be undifferentiated stem cells and derived from anysource or process which provides viable undifferentiated stem cells. Themethods described above for retrieving stem cells from embryos is mostpreferred.

In these preferred aspects, the conditions of culturing the cells athigh density on a fibroblast feeder cell layer or transferring to aserum free medium are intended to be non-permissive for stem cellrenewal or cause uni-directional differentiation toward extraembryoniclineages.

Generally the presence of a fibroblast feeder layer will maintain thesecells in an undifferentiated state. This has been found to be the casewith the cultivation of mouse and human ES cells. However, without beingrestricted by theory, it has now become evident that the type andhandling of the fibroblast feeder layer is important for maintaining thecells in an undifferentiated state or inducing differentiation of thestem cells.

Suitable fibroblast feeder layers are discussed above.

Somatic differentiation in vitro of the ES cell lines is a function ofthe period of cultivation following subculture, the density of theculture, and the fibroblast feeder cell layer. It has been found thatsomatic differentiation may be detected as early as the first week aftersubculture and is morphologically apparent and demonstrable byimmunochemistry approximately 14 days following routine subcultivationas described above in areas of the colony which are remote from directcontact with the feeder cell layer (in contrast to areas adjacent to thefeeder cell layer where rapid stem cell growth is occurring such as theperiphery of a colony at earlier time points after subcultivation), orin cultures which have reached confluence. Depending upon the method ofpreparation and handling of the mouse embryo fibroblasts, the mousestrain from which the fibroblasts are derived, and the quality of aparticular batch, stem cell renewal, extraembryonic differentiation orsomatic differentiation may be favoured.

Once a suitable fibroblast cell line is selected, it may be used as adifferentiation inducing fibroblast feeder layer to induce theundifferentiated stem cells to differentiate into a somatic lineage ormultiple somatic lineages. These may be identified using markers or geneexpression as described above. Preferably the fibroblast feeder layerdoes not induce extraembryonic differentiation and cell death.

The modulation of stem cell growth by appropriate use of fibroblastfeeder layer and manipulation of the culture conditions thus provides anexample whereby somatic differentiation may be induced in vitroconcomitant with the limitation of stem cell renewal without theinduction of widespread cell death or extraembryonic differentiation.

Other manipulations of the culture conditions such as culturing invarious compositions of serum free medium may be used to arrest stemcell renewal without causing stem cell death or unidirectionalextraembryonic differentiation, thereby favouring differentiation ofsomatic cells.

Differentiation may also be induced by culturing to a high density inmonolayer or on semi-permeable membranes so as to create structuresmimicking the postimplantation phase of human development, or anymodification of this approach. Cultivation in the presence of cell typesrepresentative of those known to modulate growth and differentiation inthe vertebrate embryo (eg. endoderm cells or cells derived from normalembyronic or neoplastic tissue) or in adult tissues (eg. bone marrowstromal preparation) may also induce differentiation, modulatedifferentiation or induce maturation of cells within specific celllineage so as to favour the establishment of particular cell lineages.

Chemical differentiation may also be used to induce differentiation.Propagation in the presence of soluble or membrane bound factors knownto modulate differentiation of vertebrate embryonic cells, such as bonemorphogenetic protein-2 or antagonists of such factors, may be used.

Applicants have found that Oct-4 is expressed in stem cells anddown-regulated during differentiation and this strongly indicates thatstem cell selection using drug resistance genes driven by the Oct-4promoter will be a useful avenue for manipulating human ES cells.Directed differentiation using growth factors, or the complementarystrategy of lineage selection coupled with growth factor enhancementcould enable the selection of populations of pure committed progenitorcells from spontaneously differentiating cells generated as describedhere.

Genetic modification of the stem cells or further modification of thosegenetically modified stem cells described above may be employed tocontrol the induction of differentiation. Genetic modification of thestem cells so as to introduce a construct containing a selectable markerunder the control of a promoter expressed only in specific celllineages, followed by treatment of the cells as described above and thesubsequent selection for cells in which that promoter is active may beused.

Once the cells have been induced to differentiate, the various celltypes, identified by means described above, may be separated andselectively cultivated. Preferably neural progenitor cells are selected.These progenitors are capable of differentiating into neuron cellsand/or glial cells. More preferably, they will differentiate into neuroncells and/or glial cells in the absence of other differentiated cellssuch as those from the extra embryonic lineage.

Selective cultivation means isolation of specific lineages ofprogenitors or mature differentiated cells from mixed populationspreferably appearing under conditions unfavourable for stem cell growthand subsequent propagation of these specific lineages. Selectivecultivation may be used to isolate populations of mature cells orpopulations of lineage specific committed progenitor cells. Isolationmay be achieved by various techniques in cell biology including thefollowing alone or in combination: microdissection; immunologicalselection by labelling with antibodies against epitopes expressed byspecific lineages of differentiated cells followed by direct isolationunder fluorescence microscopy, panning, immunomagnetic selection, orselection by flow cytometry; selective conditions favouring the growthor adhesion of specific cell lineages such as exposure to particulargrowth or extracellular matrix factors or selective cell-cell adhesion;separation on the basis of biophysical properties of the cells such asdensity; disaggregation of mixed populations of cells followed byisolation and cultivation of small clumps of cells or single cells inseparate culture vessels and selection on the basis of morphology,secretion of marker proteins, antigen expression, growth properties, orgene expression; lineage selection using lineage specific promoterconstructs driving selectable markers or other reporters.

The derivation of neural progenitors from ES cells, and even furthermore, the establishment of a pure neural progenitor cell line isdescribed below as proof of the above principles. The followingdescription is illustrative of neural progenitor cells as somatic cellsdifferentiated from stem cells and should not be taken as a restrictionon the generality of the invention. It should be noted that the methodmay be used to derive an enriched preparation of a variety of somaticprogenitors such as but not limited to mesodermal progenitors such ashemangioblast or hematopoietic stem cells or neural progenitors.Transdifferentiation to non-neuronal phenotypes from neural progenitorsis within the scope of the present invention and may result inhematopoietic, endothelial, embryonic endoderm and ectodermal cells.

The establishment of neural progenitor cells from embryonic stem cellsand more preferably a pure preparation of neural progenitor cells andeven more preferably a neural progenitor cell line may be achieved byany one or combination of the following approaches.

In one preferred approach, somatic differentiation of ES cells isinduced by prolonged culture of ES cells to high density on anappropriate fibroblast feeder layer that prevents unidirectionaldifferentiation towards extraembryonic lineage and promotes somaticdifferentiation. Once the cells have been induced to differentiatetoward somatic lineages, areas which are destined to give rise toclusters of mainly neural progenitor cells may be identified based oncharacteristic morphological features as described above. The size anddemarcation of these areas may be enhanced by replacing the growthmedium with serum free medium supplemented with EGF and bFGF. The areasare separated mechanically and replated in serum-free medium, whereuponthey form spherical structures.

Any serum free medium may be used. Preferably NS-A (Euroclone) orDMEM/F12 (Gibco) is used. More preferably NS-A or DMEM/F12 supplementedwith N2 or B27 (Gibco) is used. Most preferably DMEM/F12 supplementedwith B27 is used.

In the presence of an appropriate supplement of growth factors such asbut not limited to, EGF and basic FGF to the serum free medium, theneural progenitors may be cultivated and expanded to establish a cellline. The growth factors inhibit further differentiation of theprogenitor cells and promote their proliferation.

The culture in the serum free medium and preferably growth factors isselective and therefore limits prolonged proliferation of other types ofdifferentiated cells such as the progeny of the extraembryonic lineagethat may coexist in the culture. Therefore the cultivation in theseselective conditions may be used to establish an enriched cell line ofneural progenitors.

The progenitors may be cultured as spheres or as a monolayer.Subculturing may be conducted mechanically. Scraping is preferred topropagate monolayer cultures. However, any mechanical method such astituration or cutting may be used to subculture the spheres. Mostpreferably the spheres are sliced into smaller clumps. The progenitorsmay be expanded to produce a large number of cells.

In another preferred approach, the method involves the transfer ofundifferentiated stem cells into culture conditions that on one handdirect differentiation toward a desired somatic lineage, which is theneural lineage in this case, while on the other hand are selective andtherefore limit both the differentiation toward unwanted lineages (suchas extraembryonic lineages) as well as the survival of differentiatedcells from these lineages. Such culture conditions include the transferinto serum free media (as described above) that may be supplemented withgrowth factors including but not limited to bFGF and EGF. The serum freemedia promotes differentiation towards the neuroectodermal lineage (andpossibly other non-neural lineages such as mesoderm). The serum freemedia may limit the growth and survival of unwanted cells such as thosefrom the extraembryonic lineages.

In a further preferred embodiment of the invention, the method allowsthe establishment of a pure progenitor cell line from the desiredlineage.

Growth factors that are added to the medium may promote theproliferation and the cultivation of the desired somatic progenitorssuch as neural progenitors. The selective culture conditions furthereliminate during cultivation, unwanted differentiated cells from otherlineages such as extraembryonic lineages. The method may be used toderive a pure preparation and/or a pure cell line of a variety ofsomatic progenitors including, but not limited to, neural progenitor,mesodermal progenitors such as hemangioblast or hematopoietic stem cellsand progenitors of the endodermal lineage.

Preferably, in the derivation of an enriched cell line of neuralprogenitors, clumps of undifferentiated stem cells may be transferredinto plastic tissue culture dishes containing serum free medium. Theserum free medium induces the differentiation of the ES cells initiallytowards ectoderm and then towards the neuroectodermal lineage.

Any serum free medium may be used. Preferably NS-A medium (Euroclone) orDMEM/F12 is used. More preferably the serum free medium is supplementedwith N2 or B27 (Gibco). Most preferably the medium is DMEM/F12supplemented with B27. The clusters of undifferentiated stem cells turninto round spheres within approximately 24 hours after transfer (FIG.9).

The serum free medium may be further supplemented with basic FGF and EGFto promote proliferation of neural progenitors in an undifferentiatedstate. The progenitors may be cultivated under these conditions forprolonged periods. The selective conditions that are induced by theserum free medium and growth factors result in a gradual purificationand elimination of other differentiated cell types during cultivation.

The progenitors may be cultured as spheres or as a monolayer.Subculturing may be conducted mechanically. Scraping is preferred topropagate monolayer cultures. Any mechanical method known to the skilledaddressee such as tituration or cutting may be used to subculture thespheres. Most preferably the spheres are sliced into smaller clumps. Theprogenitors may be expanded to produce a large number of cells.

The progenitors that are generated directly from undifferentiated stemcells have similar properties to the neural progenitors that aregenerated from differentiating stem cells colonies. They express thesame markers of primitive neuroectoderm and neural progenitor cells,such as N-CAM, polysialyated N-CAM, the intermediate filament proteinnestin, Vimentin and the transcription factor Pax-6. They do not expressthe transcriptional factor oct-4. They have a similar growth potential.They generate differentiated neural cells with similar morphology andmarker expression after plating on appropriate substrate and withdrawalof growth factors.

In another aspect of the invention, there is provided a method ofinducing somatic cells from embryonic stem cell derived somaticprogenitors, said method comprising:

obtaining a source of embryonic stem cell derived somatic progenitorcells;

culturing the progenitor cells on an adhesive substrate; and

inducing the cells to differentiate to somatic cells under conditionswhich favour somatic differentiation.

The source of embryonic stem cell derived progenitor cells may be fromany source. However, they are preferably established by the methodsdescribed above. Preferably, the cells are grown in the presence of aserum-free media and growth factor.

The somatic cells may preferably be neurons, or glial cells includingastrocytes or oligodendrocyte cells. Preferably, the somatic progenitorsare neural progenitors.

Any adhesive substrate may be used. More preferably, poly-D-lysine andlaminin or poly-D-lysine and fibronectin are used.

Induction of somatic cells is preferably achieved by withdrawing growthfactors from the media. However, other acceptable methods of inductionmay be used. These may include:

culturing the undifferentiated cells for prolonged periods and at highdensity to induce differentiation;

culturing the cells in serum free media;

culturing the cells on a differentiation inducing fibroblast feederlayer and wherein said fibroblast feeder layer does not induce extraembryonic differentiation and cell death;

culturing to a high density in monolayer or on semi-permeable membraneso as to create structures mimicking the postimplantation phase of humandevelopment; or

culturing in the presence of a chemical differentiation factor selectedfrom the group including bone morphogenic protein-2 or antagoniststhereof.

For inducing neurons, it is preferred to further use poly-D-lysine andlaminin.

Upon plating of neural progenitors on an appropriate substrate such aspoly-D-lysine and laminin, and withdrawal of growth factors from theserum free medium, differentiated cells grow out of the spheres as amonolayer and acquire morphology of mature neurons and expression ofmarkers such as the 160 kDa neurofilament protein, Map-2AB,synaptophysin, Glutamate, GABA, serotonin, tyrosine hydroxylase, GABAbiosynthesis and receptor subunits characteristic of GABA minergicneurons (GABA Aα2) which are characteristic of mature neurons.

In a preferred embodiment, the method for inducing neurons furtherincludes culturing the somatic progenitor cells, preferablyundifferentiated neural progenitor cells, or differentiating neuronalprogenitors in the presence of retinoic acid.

Retinoic acid has been found to further induce differentiation towardmature neurons.

Accordingly, there is provided a mature neuron cell prepared by themethod described herein. The mature neuron cell may be characterized inthat it expresses 160 kDa neuro-filament protein, MAP2ab, glutamate,synaptophysin, glutamic acid decarboxylase (GAD), tyrosine hydroxylase,GABA and serotonin.

The establishment of oligodendrocyte and astrocyte cells indicates thepotential of the neural precursors to differentiate towards the gliallineage.

For inducing of glial cells including astrocytes and oligodendrocyteprogenitors, it is preferred to use poly-D-lysine and fibronectin.Fibronectin is significantly more potent than laminin for the inductionof differentiation towards the glial lineage.

In a preferred embodiment, the method for inducing glial cells furtherincludes culturing the somatic progenitor cells, preferablyundifferentiated neural progenitor cells, in the presence of PDGF-AA andbasic FGF.

In yet another preferred embodiment, the method for inducing glial cellsfurther includes culturing the somatic progenitor cells, preferablyundifferentiated neural progenitor cells, in the presence of T3. Thecells may be then grown in the absence of growth factor.

The glial cells may be selected from astrocytes or oligodendrocytes. Theoligodendrocytes derived from the methods of the present invention maybe identified and characterized by RNA transcripts of MBP, plp and dm-20or by immunostaining for O4 and NG2.

Culture in serum free medium supplemented with b-FGF and PDGF-AA maydirect the neural progenitors to turn into glial progenitors and inducethe expansion of glial progenitors. This is followed by plating theprogenitors on poly-D-lysine and fibronectin and further culture in thepresence of the growth factors and T3 followed by culture in thepresence of T3 without growth factor supplementation. Without beinglimited by theory, it is postulated that the growth factors such as bFGFand PDGF-AA facilitate proliferation and spreading of the glialprogenitors, fibronectin further induces differentiation towards theglial lineage and T3 induce the differentiation toward and along theoligodendrocyte lineage.

In another aspect, differentiation into glial cells including astrocyteand oligodendrocyte cells is induced by plating the neural progenitorson poly-D-lysine and fibronectin and culturing them in the serum freemedium supplemented with EGF, b-FGF and PDGF-AA. The growth factors maythen be removed and the cells further cultured in the presence of T3.

In yet another aspect, the invention provides differentiated somaticcells including neural, neural progenitor cells, neuronal and/or glialcells prepared by the methods of the present invention. The glial cellsinclude astrocytes or oligodendrocytes. The oligodendrocytes derived bythe methods of the present invention may be characterized by RNAtranscripts of MBP, plp and dm-20 or by immunostaining for O4 and NG2.

The progenitor cells that are derived by the method that is describedabove may be used to generate differentiated cells from other lineages.The spheres of progenitors may include in addition to neural progenitorsmore primitive cells such as primitive ectodermal cells or progenitorcells of other lineages such as the hemangioblast endothelial orhematopoietic stem cells, embryonic endodermal cells and ectoderm. Bymanipulation of the culture conditions these primitive cells maygenerate all somatic cell types.

Expression of mesodermal markers such as flk-1, AC-133 and CD-34,embryonic endodermal markers such as transferin, amylase and α1 antitrypsin and the epidermal marker keratin has been demonstrated in thehuman ES derived progenitor cell preparation. This may indicate thepresence of primitive cells from non-neural lineages such as thehemangioblast cell or hematopoietic stem cell within the neuralprogenitors preparation. Alternatively it may be that the primitiveneural progenitors within the spheres express these markers. Theexpression of the markers may indicate the possible high plasticity ofthe neural progenitors to transdifferentiate into cells of otherlineages.

The present invention provides a method that generates an in vitro andin vivo model of controlled differentiation of ES cells towards theneural lineage. The model, and the cells that are generated along thepathway of neural differentiation may be used for the study of thecellular and molecular biology of human neural development, for thediscovery of genes, growth factors, and differentiation factors thatplay a role in neural differentiation and regeneration. The model, andthe cells that are generated along the pathway of neural differentiationmay be used for drug discovery and for the development of screeningassays for teratogenic, toxic and neuroprotective effects.

In a further aspect of the invention, there is provided a method ofproducing large quantities of differentiated and undifferentiated cells.It is intended to mean that these cells can be propagated, expanded andgrown in cell culture.

In yet another aspect, the present invention provides a method ofproducing an enriched preparation of human ES derived neural progenitorcells, said method comprising:

obtaining an undifferentiated human embryonic stem cell as describedherein;

inducing somatic differentiation of the embryonic stem cell to a neuralprogenitor cell by a method described herein;

identifying a neural progenitor cell by expressed markers of primitiveneuroectoderm and neural stem cells such as N-CAM, polysialyated N-CAM,A2B5, intermediate filament proteins such as nestin and vimentin and thetranscription factor Pax-6; and

culturing the neural progenitor cells to promote proliferation andpropagation.

The neural progenitor cells will grow as spheres or monolayerspreferably in serum free media. A suitable media is DMEM/F12supplemented with growth factors selected from the group including B27,EGF and bFGF.

Further enrichment of the preparation may be achieved by furthercultivation in new media that includes transferring the clumps of cellsinto new media.

In a further aspect of the invention there is provided a method todis-aggregate the spheres into single cell suspensions. Dis-aggregationby using digestion with trypsin or dispase may be ineffective.Dis-aggregation may be accomplished by digestion with papain combinedwith mechanical tituration.

In another aspect of the invention, there is provided a method oftransplanting ES derived neural progenitor spheres, said methodcomprising:

disaggregating the spheres; and

injecting the disaggregated spheres into a living host.

Disaggregation of the spheres may be conducted in any way to separatethe cells either to small clumps or single cells. Ideally, trypsin ordispase are not used. Mechanical disaggregation or tituration may beadopted to separate the cells prior to injection. Alternatively thespheres may be disaggregated by digestion with papain preferablycombined with mechanical tituration.

Injection may be conducted in any manner so as to introduce the cellsinto the nervous system of the host. Preferably the cells are introducedinto a specific site in the nervous system. Any method may be used tointroduce the cells into a specific location. Preferably, the cells areinjected using a micro-glass pipette (300 micron outer diameter)connected to a micro-injector (Narishige, Japan). The glass pipette maybe covered by a plastic sleeve that will limit the depth of penetrationinto the host nervous system. The cells may be also injected by ahamilton syringe into predetermined depth using a stereotaxic device.Any stereotaxic injection method may be suitable.

The volume that is injected and the concentration of cells in thetransplanted solution depend on the indication for transplantation, thelocation in the nervous system and the species of the host. Preferably 2microliters with 25,000-50,000 cells per microliter are injected to thelateral cerebral ventricles of newborn rats or mice.

In another aspect of the invention there is provided a neural progenitorcell capable of transplantation into a host nervous system said cellcharacterised by establishing a stable graft and contributing in thehistogenesis of a living host.

In another aspect of the present invention there is provided a method ofinducing somatic cells in vivo from embryonic stem cell derived somaticprogenitors, said method comprising:

obtaining a source of embryonic stem cell derived somatic progenitorcells, preferably prepared by the methods described herein; and

transplanting the somatic progenitors into a host to inducedifferentiation to somatic cells.

The transplanting may be conducted by any of the methods describedherein.

When engrafted into a developing nervous system, the progenitor cells ofthe present invention will participate in the processes of normaldevelopment and will respond to the host's developmental cues. Theengrafted progenitor cells will migrate along established migratorypathways and will spread widely into disseminated areas of the nervoussystem. The transplanted cells will respond to host environmentalsignals, differentiate in a temporally and regionally appropriate mannerinto progeny from both the neuronal and glial lineages in accord to theregion's stage of development and in concert with the host developmentalprogram. The engrafted neural progenitor cell is capable ofnon-disruptive intermingling with the host neural progenitors as well asdifferentiated cells. The transplanted cells can replace specificdeficient neuronal or glial cell populations, restore defectivefunctions and can express foreign genes in a wide distribution.

In a further aspect of the invention the ES derived neural progenitorcells or their differentiated progeny may be transplanted into thedeveloped nervous system. They can form a stable graft, migrate withinthe host nervous system, intermingle and interact with the host neuralprogenitors and differentiated cells. They can replace specificdeficient neuronal or glial cell populations, restore deficientfunctions and activate regenerative and healing processes in the host'snervous system. In an even further aspect of the invention thetransplanted cells can express foreign genes in the host's nervoussystem.

Preferably the stable graft is a graft established in the centralnervous system or the peripheral nervous system. The stable graft mayestablish in the brain. The neural progenitor cells of the presentinvention have been shown by the applicants to differentiate to matureneurons characterized by their ability to express 160 kDa neuro-filamentprotein, MAP2ab, glutamate, synaptophysin, glutamic acid decarboxylase(GAD), tyrosine hydroxylase, GABA and serotonin. The neural progenitorcells of the present invention have been also shown by the applicants todifferentiate to astrocyte and oligodendrocyte characterized by RNAtranscripts of GFAP, MBP, plp and dm-20 or by immunostaining for GFAP,O4 and NG2.

The ability to differentiate to the various somatic cell types isparticularly useful for modifying the nervous system for replacingdeficient neuronal or glial cell populations, restoring deficientfunctions of the system, or activating regenerative and healingprocesses in the nervous system.

In a further aspect of the invention the progenitor cells are graftedinto other organs such as but not limited to the hematopoietic systemwhere they trans-differentiate and form a stable functional graft.

More preferably the spheres are ES derived human neural progenitorspheres which are transplanted into the living host.

In yet another aspect of the present invention, there is provided amethod of treating a mental condition by replenishing a cell populationin the brain, said method comprising:

obtaining a source of embryonic stem cell derived somatic progenitorcells, preferably prepared by the methods described herein; and

transplanting the somatic progenitors into a host to promote theirdifferentiation to somatic cells.

Preferably, they are differentiated from neural lineages selected fromneurons, astrocytes and oligodendrocyte.

Preferably the mental condition is selected from the group includingalzheimers disease, and other mental conditions causing dementia. It isshown that mature neurons of the present invention express glutamate,TH, GABA and serotonin. Serotoninergic neurons may have a role in thepathogenesis and treatment of mental conditions.

In a further aspect of the invention there is provided a neuralprogenitor cell, a neuronal cell and/or a glial cell that may be usedfor cell therapy in a variety of pathological conditions including butnot limited to neurodegenerative disorders, vascular conditions,autoimmune disorders, congenital disorders, trauma and others.

In a further aspect of the invention there is provided a neuralprogenitor cell, a neuronal cell and/or a glial cell that may be usedfor gene therapy. Genetically manipulated neural progenitor cells orneuronal cell or glial cells may be used after transplantation as avector to carry and express desired genes at target organs.

In another aspect of the present invention, there is provided acommitted progenitor cell line. The progenitor cell line may be expandedto produce large quantities of progenitor cells, neural progenitorcells, neuronal cells, mature neuronal cells and glial cells.

In another aspect of the invention, there are provided committed neuralprogenitor cells capable of self renewal or differentiation into one orlimited number of somatic cell lineages, as well as maturedifferentiated cell produced by the methods of the present invention.

Expansion of the committed progenitor cells may be useful when thenumber of progenitors that may be derived from ES cells is limited. Insuch a case, expansion of the progenitors may be useful for variousapplications such as the production of sufficient cells fortransplantation therapy, for the production of sufficient RNA for genediscovery studies etc. For example, by using the techniques describedabove, expansion of progenitor cells from ten spheres for ten passagesmay generate 50×10⁶ cells that would be sufficient for any application.

These observations on cells of the neural lineage establish theprinciple that by using the techniques described, committed progenitorcells may be isolated, from embryonic stem cell cultures propagated,expanded, enriched and further induced to produce fully differentiatedcells.

In a further aspect of the invention, there is provided a method ofproducing large quantities of differentiated and undifferentiated cells.

In another aspect there is provided a differentiated committedprogenitor cell line that may be cultivated for prolonged periods andgive rise to large quantities of progenitor cells and fullydifferentiated cells.

The neural progenitor cells or other committed progenitor cells derivedby the method described above may be used to generate differentiatedcells from other lineages by transdifferentiation.

In another aspect there is provided a differentiated committedprogenitor cell line capable of differentiation into mature neuronsand/or glial cells. Preferably the progenitor cell is a neuralprogenitor cell.

In another aspect there is provided an undifferentiated cell linecapable of differentiation into neural progenitor cells produced by themethod of the present invention.

Specific cell lines HES-1 and HES-2 were isolated by the proceduresdescribed above and have the properties described above.

In another aspect of the invention there is provided a cell compositionincluding a human differentiated or undifferentiated cell capable ofdifferentiation into neural progenitor cells preferably produced by themethod of the present invention, and a carrier.

The carrier may be any physiologically acceptable carrier that maintainsthe cells. It may be PBS or ES medium.

The differentiated or undifferentiated cells may be preserved ormaintained by any methods suitable for storage of biological material.Vitrification of the biological material is the preferred method overthe traditional slow-rate freezing methods.

Effective preservation of ES cells is highly important as it allows forcontinued storage of the cells for multiple future usage. Althoughtraditional slow freezing methods, commonly utilised for thecryo-preservation of cell lines, may be used to cryo-preserveundifferentiated or differentiated cells, the efficiency of recovery ofviable human undifferentiated ES cells with such methods is extremelylow. ES cell lines differ from other cell lines since the pluripotentcells are derived from the blastocyst and retain their embryonicproperties in culture. Therefore, cryo-preservation using a method whichis efficient for embryos is most appropriate. Any method which isefficient for cryo-preservation of embryos may be used. Preferably,vitrification method is used. More preferably the Open Pulled Straw(OPS) vitrification method previously described by Vajta, G. et al(1998) Molecular Reproduction and Development, 51, 53-58, is used forcryopreserving the undifferentiated cells. More preferably, the methoddescribed by Vajta, G. et al (1998) Cryo-Letters, 19, 389-392 isemployed. Generally, this method has only been used for cryopreservingembryos.

The committed progenitor cell line is efficiently recovered fromcryopreservation using the traditional slow rate cooling method.

The differentiated or undifferentiated cells may be used as a source forisolation or identification of novel gene products including but notlimited to growth factors, differentiation factors or factorscontrolling tissue regeneration, or they may be used for the generationof antibodies against novel epitopes. The cell lines may also be usedfor the development of means to diagnose, prevent or treat congenitaldiseases.

Much attention recently has been devoted to the potential applicationsof stem cells in biology and medicine. The properties ofpluripotentiality and immortality are unique to ES cells and enableinvestigators to approach many issues in human biology and medicine forthe first time. ES cells potentially can address the shortage of donortissue for use in transplantation procedures, particularly where noalternative culture system can support growth of the required committedstem cell. ES cells have many other far reaching applications in humanmedicine, in areas such as embryological research, functional genomics,identification of novel growth factors, and drug discovery, andtoxicology.

While the potential applications of neural stem cells derived from adultor embryonic CNS are considerable, there may be real advantages toneural progenitor cells derived from ES cell cultures.

ES cell lines derived from a patients' own tissue via somatic cellnuclear transfer would produce neuronal precursors which are a precisematch to the recipients own tissue and might therefore be more suitablefor grafting.

Moreover the use of nuclear transfer to yield ES cells from individualswith specific genetic predispostions to certain diseases of the CNScould provide a powerful tool for the generation of in vitro models fordisease pathogenesis.

It is quite likely that neural precursors generated from ES cellcultures may demonstrate a greater growth or developmental potentialthan committed progenitors from fetal or adult CNS.

There is a huge range of cell types within the adult CNS, and while itis clear that ES cells can give rise to any of these in the mouse, it isnot clear that neural stem cells can do so.

ES derived neural progenitors may allow the study of early stages of theprocess of neurogenesis, and thereby provide important clues fordiscovery of novel factors enhancing tissue regeneration, or novel stemcell intermediates which might be more facile at replacing damagedtissue.

It may be that the frequency of homologous recombination in ES cells ismuch higher than that in neural stem cells, and therefore that the onlypractical route for introducing targetted genetic modifications intohuman neural tissue-either for generation of disease models in vitro orfor types of gene therapy-lies in the reproducible generation andisolation of neural progenitors from genetically modified embryonic stemcells.

The present invention will now be more fully described with reference tothe following examples. It should be understood, however, that thedescription following is illustrative only and should not be taken inany way as a restriction on the generality of the invention describedabove.

REFERENCES

-   1. Evans, M. J. and Kaufman, M. Establishment in culture of    pluripotential stem cells from mouse embryos. Nature 292, 151-156    (1981).-   2. Martin, G. R. Isolation of a pluripotent cell line from early    mouse embryos cultured in medium conditioned by teratocarcinoma stem    cells. Proc. Natl. Acad. Sci. U.S.A. 78, 7634-7638 (1981).-   3. Andrews, P. W. et al. Pluripotent embryonal carcinoma clones    derived from the human teratocarcinoma cell line Tera-2. Lab.    Invest. 50, 147-162 (1984).-   4. Pera, M. F., Cooper, S., Mills, J., & Parrington, J. M. Isolation    and characterization of a multipotent clone of human embryonal    carcinoma-cells. Differentiation 42, 10-23 (1989).-   5. Thomson, J. A. et al. Isolation of a primate embryonic stem cell    line. Proc. Natl. Acad. Sci. U.S.A. 92, 7844-7844 (1995).-   6. Thomson, J. A. et al. Pluripotent cell lines derived from common    marmoset (Callithrix jacchus) blastocysts. Biol. Reprod. 55,    254-259. (1996).-   7. Bongso A., Fong C. Y., Ng S. C., and Ratnam, S. Isolation and    culture of inner cell mass cells from human blastocysts. Hum.    Reprod. 9, 2110-2117 (1994).-   8. Thomson, J. A. et al. Embryonic stem cell lines derived from    human blastocysts. Science 282, 1145-1147 (1998).-   9. Andrews, P. W. et al. Comparative-analysis of cell-surface    antigens expressed by cell-lines derived from human germ-cell    tumors. Int. J. Cancer 66, 806-816 (1996).-   10. Cooper, S., Pera, M. F., Bennett, W., & Finch, J. T. A novel    keratan sulfate proteoglycan from a human embryonal carcinoma    cell-line. Biochem. J. 286, 959-966 (1992).-   11. Pera, M. F. et al. Analysis of cell-differentiation lineage in    human teratomas using new monoclonal-antibodies to cytostructural    antigens of embryonal carcinoma-cells. Differentiation 39, 139-149    (1988).-   12. Fong C. Y., and Bongso A. Comparison of human blastulation rates    and total cell number in sequential culture media with and without    co-culture. Hum. Reprod. 14, 774-781 (1999).-   13. Fong C. Y. et al. Ongoing pregnancy after transfer of zona-free    blastocysts: implications for embryo transfer in the human. Hum.    Reprod. 12, 557-560 (1997).-   14. Solter D., and Knowles, B. Immunosurgery of mouse blastocyst.    Proc. Natl. Acad. Sci. U.S.A. 72, 5099-5102 (1975).-   15. Vajta G, Holm P, Kuwayama M, Both P J, Jacobsen H, Greve T,    Callesen H. Open pulled straw (OPS) vitrification: A new way to    reduce cryoinjuries of bovine ova and embryos. Molecular    Reproduction and Development 1998, 51: 53-58.-   16. Vajta G, Lewis I M, Kuwayama M, Greve T, Callesen H. Sterile    application of the opened pulled straw (OPS) vitrification method.    Cryo-Letters 1998, 19: 389-392.-   17. Beddington, R. S. P. & Robertson, E. J. Axis development and    early asymmetry in mammals. Cell 96, 195-209 (1999).-   18. Li, M., Pevny, L., Lovell-Badge, R., and Smith, A. Generation of    purified neural precursors from embryonic stem cells by lineage    selection. Curr. Biol. 8, 971-974 (1998).-   19. Svensden, C N and Smith AG New prospects for human stem-cell    therapy in the nervous system. Trends in Neurosci 22: 357, 1999.-   20. Kleinsmith L J and Pierce GB. Multipotentiality of single    embryonal carcinoma cells. Cancer Res. 1964; 24: 797-842.-   21. Stevens (1983)-   22. Martin G R. Teratocarcinomas and mammalian embryogenesis.    Science 1980; 209: 768.-   23. Matsui Y, Zsebo K, Hogan B L M. Derivation of pluripotent    embryonic stem cells from murine primordial germ cells in culture.    Cell 1992; 70: 841-847.-   24. Andrews P W. Human teratocarcinoma. Biochim. Biophys. Acta 1988;    948: 17-36.-   25. Thompson S, Stern P L, Webb M, et al. Cloned human teratoma    cells differentiate into neuron-like cells and other cell types in    retinoic acid. J Cell Sci. 1984; 72: 37-64.-   26. van Eijk M J T, van Rooijen M A, Modina S, Scesi L, Folkers G,    van Tol H T A, Bevers M M, Fisher S R, Lewin H A, Shehu D, Galli C,    de Vaureix C, Trounson A O, Mummery C L, and Gandolfi F. Molecular    cloning, genetic mapping and developmental expression of bovine    POU5F1. Biology of Reproduction 1999; 60: 1093-1103.-   27. Kukekov V G, Laywell E D, Suslov O et al. Multipotent    stem/progenitor cells with similar properties arise from two    neurogenic regions of adult human brain. Experimental Neurology    1999, 156: 333-344-   28. Uchida N, Buck D W, He D, Reitsma M J, Masek M, Phan T V,    Tsukamoto A S, Gage F H, Weissman I L. Direct isolation of human    central nervous system stem cells. Proc Natl Acad Sci USA 2000,    97:14720-5.-   29. Clarke D L, Johansson C B, Wilbertz J, Veress B, Nilsson E,    Karlstrom H, Lendahl U, Frisen J. Generalized potential of adult    neural stem cells. Science 2000, 288: 1660-3.-   30. Bjornson C R, Rietze R L, Reynolds B A, Magli M C, Vescovi A L.    Turning brain into blood: a hematopoietic fate adopted by adult    neural stem cells in vivo. Science 1999, 283: 534-7.-   31. Vescovi, A. L. et al. Isolation and cloning of multipotential    stem cells from the embryonic human CNS and establishment of    transplantable human neural stem cell lines by epigenetic    stimulation. Exp. Neurol. 156, 71-83 (1999).-   32. Neelands, T. R. et al. GABA_(A) receptor pharmacology and    subtype expression in human neuronal NT2-N cells. J. Neurosci. 18,    4993-5007 (1998).-   33. Reubinoff B E, Pera M F, Fong C Y, Trounson A and Bongso A.    Embryonic stem cells. PCT/AU99/00990, U.S. application Ser. No.    09/436,164.-   34. Neelands, T. R et al GABA_(A) receptor pharmacology and subtype    expression in human neuronal NT2 N cells. J Neurosol 18, 4993-5007    (1998).-   35. Reubinoff B E, Pera M F, Fong C Y, Trounson A and Bongso A.    Embryonic stem cells. PCT/AU99/00990, U.S. application Ser. No.    09/436,164.

EXPERIMENTAL PROTOCOLS 1. Derivation and Propagation of ES Cells.

Fertilised oocytes were cultured to the blastocyst stage (day 6 afterinsemination), in sequential media, according to a standard co-culturefree protocol (Fong C. Y., and Bongso A. Comparison of humanblastulation rates and total cell number in sequential culture mediawith and without co-culture. Hum. Reprod. 14, 774-781 (1999)). Afterzona pellucida digestion by pronase (Sigma, St. Louis, Mo.)(Fong C. Y.et al. Ongoing pregnancy after transfer of zona-free blastocysts:implications for embryo transfer in the human. Hum. Reprod. 12, 557-560(1997)), ICM were isolated by immunosurgery (Solter D., and Knowles, B.Immunosurgery of mouse blastocyst. Proc. Natl. Acad. Sci. U.S.A. 72,5099-5102 (1975)) using anti-human serum antibody (Sigma) followed byexposure to guinea pig complement (Life Technologies, Gaithersburg,Md.). ICM were then cultured on mitomycin C mitotically inactivatedmouse embryonic fibroblast feeder layer (75,000 cells/cm2) in gelatinecoated tissue culture dishes. The culture medium consisted of DMEM(Gibco, without sodium pyruvate, glucose 4500 mg/L) supplemented with20% fetal bovine serum (Hyclone, Logan, Utah), 0.1 mMbeta-mercaptoethanol, 1% non essential amino acids, 2 mM glutamine, 50u/ml penicillin and 50 (g/ml streptomycin (Life Technologies). Duringthe isolation and early stages of ES cell cultivation, the medium wassupplemented with human recombinant leukemia inhibitory factor hLIF at2000 u/ml (Amrad, Melbourne, Australia). 6-8 days after initial plating,ICM like clumps were removed mechanically by a micropipette fromdifferentiated cell outgrowths and replated on fresh feeder layer. Theresulting colonies were further propagated in clumps of about 100 stemcell like cells, on mouse feeder layer, about every 7 days. The clumpswere either dissociated mechanically, or with a combined approach ofmechanical slicing followed by exposure to dispase (10 mg/ml, LifeTechnologies).

(a) Embryo Culture

Following insemination, embryos were cultured in droplets underpre-equilibrated sterile mineral oil in IVF-50 medium (Scandinavian 2medium) for 2 days.

A mixture 1:1 of IVF-50 and Scandinavian 2 medium (Scandinavian 2medium) was used in the third day.

From the forth day of culture, only Scandinavian 2 medium was used togrow the cleavage stage embryos to blastocysts.

(b) Zona Pellucida Digestion.

Zona pellucida digestion was performed at the expanded blastocyst stageon day 6.

The digestion solution included Pronase (Sigma, TC tested) 10 u in PBSand Scandinavian 2 medium (1:1).

The embryos were incubated in pronase solution for 1-1.5 min, washed inScandinavian 2 medium and incubated for 30 minutes. If the zona was notcompletely dissolved, the embryos were further incubated in pronasesolution for 15 seconds.

(c) Human Stem Cell Culture.

Human stem cells were grown on MMC treated fibroblasts' feeder layer.Fibroblasts were plated on gelatine treated dishes. A combination ofhuman and mouse derived fibroblasts were used at a density ofapproximately 25,000 and 70,000 cells per cm² respectively. Thefibroblasts were plated up to 48 hours before culture of the stem cells.Mouse fibroblasts only could also support the growth of the stem cells.However, while human fibroblasts could also support stem cells, theycreated an uneven and unstable feeder layer. Therefore, the humanfibroblasts were combined with the mouse fibroblasts to augment andachieve better support of growth and prevention of differentiation.

The medium that was used for the growth of human stem was DMEM (GIBCO,without sodium pyruvate, with glucose 4500 mg/L) supplemented with 20%FBS (Hyclone, Utah) (-mercaptoethanol—0.1 mM (GIBCO), Non EssentialAmino Acids—NEAA 1% (GIBCO), glutamine 2 mM. (GIBCO), penicillin 50u/ml, and streptomycin 50 (g/ml (GIBCO). At the initial isolation of thestem cells the medium was supplemented by hLIF 2000 u/ml. It was latershown that LIF was not necessary.

(d) Human Stem Cell Propagation

Following plating, the isolated ICM attached and was cultured for 6days. At that stage, a colony which included a clump of stem cells ontop of differentiated cells developed. The ICM clump was isolated andremoved mechanically by a micro-pipette with the aid of using Ca/Mg freePBS medium to reduce cell to cell attachments.

The isolated clump was replated on fresh human/mouse fibroblast feederlayer. Following 2 weeks of culture, a colony with typical morphology ofprimate pluripotent stem cells developed. The stem cells were furtherpropagated in one of two methods. In both methods cells which appearednondifferentiated were propagated in clumps of about 100 cells every 5-7days.

In the first method, Ca²⁺/Mg²⁺ free PBS medium was used to reduce cellto cell attachments. Following about 15-20 minutes, cells graduallystart to dissociate and the desired size clumps can be isolated. Whencell dissociation is partial, mechanical dissociation using the sharpedge of the pipette assisted with cutting and the isolation of theclumps.

An alternative approach was performed by the combined use of mechanicalcutting of the colonies followed by isolation of the subcolonies bydispase. Cutting of the colonies was performed in PBS containing Ca andMg. The sharp edge of micropipette was used to cut the colonies toclumps of about 100 cells. The pipette was also used to scrape andremove differentiated areas of the colonies. The PBS was then changed toregular prequilibrated human stem cells medium containing dispase(Gibco) 10 mg/ml and incubated for 5-10 minutes (at 37 (C, 5% CO2). Assoon as the clumps were detached they were picked up by wide boremicro-pipette, washed in PBS containing Ca and Mg and transferred to afresh feeder layer.

e) Human Stem Cell Cryopreservation.

Early passage cells were cryo-preserved in clumps of about 100 cells byusing the open pulled straw (OPS) vitrification method (Vajta et al1998) with some modifications. French mini-straws (250 (l, IMV, L'Aigle,France) were heat-softened over a hot plate, and pulled manually untilthe inner diameter was reduced to about half of the original diameter.The straws were allowed to cool to room temperature and were than cut atthe narrowest point with a razor blade. The straws were sterilised bygamma irradiation (15-25 K Gy). Two vitrification solutions (VS) wereused. Both were based on a holding medium (HM) which included DMEMcontaining HEPES buffer (Gibco, without sodium pyruvate, glucose 4500mg/L) supplemented with 20% fetal bovine serum (Hyclone, Logan, Utah).The first VS (VS1) included 10% dimethyl sulfoxide (DMSO, Sigma) and 10%ethylene glycol (EG, Sigma). The second vitrification solution (VS2)included 20% DMSO, 20% EG and 0.5M sucrose. All procedures wereperformed on a heating stage at 37 (C. 4-6 clumps of ES cells were firstincubated in VS 1 for 1 minute followed by incubation in VS2 for 25seconds. They were then washed in a 20 (l droplet of VS2 and placedwithin a droplet of 1-2 (l of VS2. The clumps were loaded into thenarrow end of the straw from the droplet by capillary action. The narrowend was immediately submerged into liquid nitrogen. Straws were storedin liquid nitrogen. Thawing was also performed on a heating stage at 37°C. as previously described with slight modifications (Vajta et al 1998).Three seconds after removal from liquid nitrogen, the narrow end of thestraw was submerged into HM supplemented with 0.2M sucrose. After 1minute incubation the clumps were further incubated 5 minutes in HM with0.1M sucrose and an additional 5 minutes in HM.

2. Stem Cell Characterisation.

Colonies were fixed in the culture dishes by 100% ethanol forimmuno-fluorescence demonstration of the stem cell surface markersGCTM-2, TRA 1-60 and SSEA-1, while 90% acetone fixation was used forSSEA-4. The sources of the monoclonal antibodies used for the detectionof the markers were as follows: GCTM-2, this laboratory; TRA 1-60, agift of Peter Andrews, University of Sheffield; SSEA-1 (MC-480) andSSEA-4 (MC-813-70), Developmental Studies Hybridoma Bank, Iowa, Iowa.Antibody localisation was performed by using rabbit anti-mouseimmunoglobulins conjugated to fluorescein isothiocyanate (Dako,Carpinteria, Calif.).

Alkaline phosphatase activity was demonstrated as previously described(Buehr M. and Mclaren A. Isolation and culture of primordial germ cells.Methods Enzymol. 225, 58-76, (1993)). Standard G-banding techniques wereused for karyotyping.

3. Oct-4 Expression Studies.

To monitor expression of Oct-4, RT-PCR was carried out on coloniesconsisting predominantly of stem cells, or colonies which had undergonespontaneous differentiation as described below. mRNA was isolated onmagnetic beads (Dynal A S, Oslo) following cell lysis according to themanufacturer's instructions, and solid-phase first strand cDNA synthesiswas performed using Superscript II reverse transcriptase (LifeTechnologies). The PCR reaction was carried out according to van Eijk etal. (1999), using the solid phase cDNA as template and Taq polymerase(Pharmacia Biotech, Hong Kong). OCT-4 transcripts were assayed using thefollowing primers: 5′-CGTTCTCTTTGGAAAGGTGTTC (forward) and3′-ACACTCGGACCACGTCTTTC (reverse). As a control for mRNA quality,beta-actin transcripts were assayed using the same RT-PCR and thefollowing primers: 5′-CGCACCACTGGCATTGTCAT-3′ (forward),5′-TTCTCCTTGATGTCACGCAC-3′ (reverse). Products were analysed on a 1.5%agarose gel and visualised by ethidium bromide staining.

4. In-Vitro Differentiation.

Colonies were cultured on mitotically inactivated mouse embryonicfibroblasts to confluency (about 3 weeks) and further on up to 7 weeksafter passage. The medium was replaced every day. Alphafetoprotein andbeta human chorionic gonadotropin levels were measured in mediumconditioned by HES-1 and HES-2 at passage level 17 and 6 respectively.After 4-5 weeks of culture, conditioned medium was harvested 36 hoursafter last medium change, and the protein levels were determined by aspecific immunoenzymometric assays (Eurogenetics, Tessenderllo, Belgium)and a fluorometric enzyme immunoassay (Dade, Miami, Fla.) respectively.These compounds were not detected in control medium conditioned only byfeeder layer.

Differentiated cultures were fixed 6-7 weeks after passage (26-HES-1 and9-HES-2) for immunofluorescence detection of lineage specific markers.After fixation with 100% ethanol, specific monoclonal antibodies wereused to detect the 68 kDa neurofilament protein (Amersham, AmershamU.K), and neural cell adhesion molecule (Dako). Muscle specific actinand desmin were also detected by monoclonal antibodies (Dako) afterfixation with methanol/acetone (1:1). Antibody localisation wasperformed as described above.

5. Teratoma Formation in Severe Combined Immunodeficient (SCID) Mice.

At the time of routine passage, clumps of about 200 cells with anundifferentiated morphology were harvested as described above, andinjected into the testis of 4-8 week old SCID mice (CB 17 strain fromthe Walter and Eliza Hall Institute, Melbourne, Australia, 10-15clumps/testis). 6-7 weeks later, the resulting tumours were fixed inneutral buffered formalin 10%, embedded in paraffin and examinedhistologically after hematoxylin and eosin staining.

6. Derivation and Culture of Neural Progenitors.

Two approaches were developed for the derivation of neural precursorsfrom human ES cells:

(a) Derivation of Neural Precursors from Differentiating ES Cells:

Colonies of undifferentiated ES cells were continuously cultured onmouse embryonic fibroblasts for 2-3 weeks. The medium was changed everyday. Starting from the second week of culture and more commonly at thethird week, areas of tight small differentiated ES cells could beidentified in the colonies both by phase contrast microscopy as well asstereo microscopy. These areas tended to become well demarcated in thethird week of culture and had a typical uniform white gray opaqueappearance under dark field stereo-microscopy (FIG. 26). The size anddemarcation of these areas could be enhanced if after the first week ofculture, the serum containing medium was replaced with serum free mediumthat was supplemented with epidermal growth factor 20 ng/ml (EGF,Gibco), and basic fibroblast growth factor 20 ng/ml (bFGF, Gibco) andconsisted of DMEM/F12 (Gibco, Gaithersburg, Md.), B27 supplementation(1:50, Gibco), glutamine 2 mM (Gibco), penicillin 50 u/ml andstreptomycin 50 μg/ml (Gibco). Clumps of about 150 small tightly packedcells were dissected mechanically by a micropipette from these areas andwere transferred to plastic tissue culture dishes containing fresh serumfree medium (as detailed above), supplemented with EGF (20 ng/ml), andbasic FGF (20 ng/ml). The medium was supplemented with heparin 5 μg/ml(Sigma St. Louis, Mo.) in some of the experiments. The clusters of cellsturned into round spheres that were comprised of small tight cellswithin 24 hours after transfer. The spheres were sub-cultured aboutevery 7-21 days. The timing of subculture was determined according tothe size of the spheres. The diameter of spheres at the time ofsub-culture was usually above 0.5 mm. Each sphere was dissectedaccording to its size to 4 parts by two surgical blades (size 20,Swann-Morton, Sheffield, UK) to produce clumps with a maximal diameterbetween 0.3-0.5 mm. 50% of the medium was changed about every 3 days.

(b) Derivation of Neural Precursors from Undifferentiated ES Cells:

Colonies of undifferentiated ES cells were propagated on mouse embryonicfibroblasts as described above. Undifferentiated ES cells were passagedin clumps of about 150-200 cells every 7 days. At the time of routinepassage, clumps of about 200 ES cells were transferred to plastic tissueculture dishes containing the same serum free medium that was describedin item 1 above. The clusters of cells turned into round spheres within24 hours after transfer. The spheres were sub-cultured about every 7-21days as described above. 50% of the medium was changed about every 3days.

(c) Characterization of the Growth and the Number of Cells in theSpheres.

Growth of the progenitors was roughly evaluated by the increase in thenumber of spheres at each passage. The growth was also monitored byserial measurements of the volume of 24 spheres. Individual spheres wereplated in twenty four well dishes (a sphere per well) and their diameterwas evaluated every 7 days starting from the first passage (one weekafter derivation). The volume was calculated by using the volumeequation of a ball. The spheres were passaged every 7-21 days when thediameter of at least six spheres exceeded 0.5 mm. At each passage, sixspheres (diameter >0.5 mm) were sectioned into quarters that were platedindividually in a 24 well tissue culture dish. When the measurementsoccurred 7 days after passage, the sum of volumes of the daughterspheres was compared to the sum of volumes of the mother spheres.

The number of cells per sphere and its correlation with the diameter ofthe spheres was evaluated in a sample of spheres with various sizes.Each sphere was mechanically disaggregated into single cells or byenzymatic (papain, Wortinington Biochemical Co, NJ) digestion that wasfollowed by tituration. The cells were than spun down re-suspended inserum free medium and counted. The cells were also stained with trypanblue to determine the rate of viable cells.

(d) Cryopreservation of Spheres.

Spheres of precursors were transferred into a 1.2 ml cryo-vial (NalgeNunc Napervville, Ill.) containing 0.5-1 ml of pre-cooled (4° C.)freezing medium (90% serum free medium (as above) and 10% DMSO (Sigma)).The vials were slowly cooled (˜1° C./min) in a freezing container(Nalgene, Nalge Nunc Napervville, Ill.) to −80° C. and then plunged intoand stored in liquid nitrogen. The vials were rapidly thawed in a waterbath at 37° C. The freezing medium was gradually diluted with 10 mlserum free culture medium and the spheres were transferred to freshserum free medium.

7. Characterization of the Progenitor Cells in the Spheres. (a)Immunohistochemistry Studies

In general, for the immunophenotyping of spheres, disaggregatedprogenitor cells and differentiated cells, fixation with 4%paraformaldehyde for 20 minutes at room temperature was used unlessotherwise specified. It was followed by blocking and permeabilizationwith 0.2% Triton X (Sigma) and 5% heat inactivated goat serum (Dako) inPBS for one hour. Samples were incubated with the primary antibodies atroom temperature for 30 minutes, washed, incubated with the secondaryantibodies for the same time, counter-stained and mounted withVectashield mounting solution with DAPI (Vector Laboratories,Burlingame, Calif.). Primary antibodies localisation was performed byusing swine anti-rabbit and goat anti-mouse immunoglobulins conjugatedto fluorescein isothiocyanate (Dako; 1:20), and goat anti mouse IgMconjugated to Texas Red (Jackson Lab. West Grove, Pa.: 1:50). Propercontrols for primary and secondary antibodies revealed neithernon-specific staining nor antibody cross reactivity.

The spheres were plated on coverslips coated with poly-D-lysine (30-70kDa, Sigma) and laminin (Sigma), fixed after 4 hours and examined byindirect immunofluorescence analysis for the expression of N-CAM(acetone fixation without permeabilization, mouse monoclonal antibodyUJ13a from Dako, Carpinteria, Calif.; 1:10 and anti polysialylatedN-CAM, clone MenB, kind gift of G. Rougon; 1:50)), A2B5 (4%paraformaldehyde fixation, mouse monoclonal antibody clone 105 fromATCC, 1:20), nestin (4% paraformaldehyde fixation, rabbit antiserum akind gift of Dr. Ron McKay; 1:25) and vimentin (methanol fixationwithout permeabilization, mouse monoclonal antibody Vim3B4 from RocheDiagnostics Australia, Castle Hill, NSW; 1:20).

To evaluate the proportion of cells that expressed N-CAM, A2B5, nestinand vimentin, spheres that were cultivated 6-18 weeks weredissaggregated into single cells either by mechanical tituration in PBSwithout calcium and magnesium or by enzymatic (papain, WortiningtonBiochemical Co, NJ) digestion that was followed by tituration. The cellswere than plated on coverslips coated with poly-D-lysine and lamininfixed after 24 hours and examined by indirect immunofluorescenceanalysis for expression of N-CAM A2B5, nestin and vimentin. DAPIcounterstain of the cell nuclei assisted in identifying individualcells. Two hundred cells were scored within random fields (at ×400) forthe expression of each marker and the scoring was repeated at least 3times for each marker. Three progenitor cell lines derived fromdifferentiating colonies and two lines that were derived directly fromundifferentiated cells were evaluated.

To examine the expression of endodermal markers, spheres were plated oncoverslips coated with poly-D-lysine and fibronectin (Sigma, 5 mcg/mk),cultured 4 weeks in the absence of growth factors and examined byindirect immunofluorescence analysis for the expression of low molecularweight (LMW) cytokeratin (4% paraformaldehyde fixation, mouse monoclonalantibody from Beckton Dickinson, San Jose, Calif.) and laminin (4%paraformaldehyde fixation, mouse monoclonal antibody, I:500 dilution,from Sigma).

(b) RT-PCR

Rt PCR was used to study the expression of nestin, the transcriptionfactor PAX-6, oct4, CD-34, FLK-1, AC-133, ultra high sulfur keratin,amylase, α1 anti trypsin, transferrin HNF-3α, and alfafetoprotein (AFP),in the spheres.

Expression of the endodermal markers HNF-3α, AFP and transferin was alsostudied in differentiated spheres that were plated on poly-D-lysine(30-70 kDa) and Fibronectin (Sigma, 5 mcg/mk) or laminin (Sigma),cultured in the same serum free medium supplemented with growth factorsfor two weeks and then further cultured two weeks without growth factorssupplementation.

The mRNA was isolated on magnetic beads (Dynal A S, Oslo) following celllysis according to the manufacturer's instructions, and solid-phasefirst strand cDNA synthesis was performed using Superscript II reversetranscriptase (Gibco, Gaithersburg, Md.).

Alternatively, total RNA was isolated by using the RNA STAT-60™ kit(Tel-Test Inc, Friendswood, Tx) and first strand cDNA synthesis wasperformed using Superscript II reverse transcriptase or SuperScriptFirst Strand Synthesis System (Gibco, Gaithersburg, Md.) according tothe manufacturers' instructions.

The PCR reaction was carried out according to van Eijk et al. (1999),using the solid phase cDNA as template and Taq polymerase (PharmaciaBiotech, Hong Kong). Alternatively, the PCR reaction mixture contained1×PCR buffer, each primer at 0.2 μM, 0.2 μM dNTPs, 1 u Taq DNAPolymerase (Gibco) or 1 u Tfl DNA Polymerase (Promega, Madison, Wis.)and 1.5 μM Mg⁺² in a final volume of 25 μl. As a control for mRNAquality, beta-actin transcripts were assayed using the same RT-PCR. PCRprimers were synthesized by Besatec or Pacific Oligos (Adelaide,Australia). The following primers were used:

Product Gene Primers size PAX-6 Forward: 5′AACAGACACAGCCCTCACAAACA3′(SEQ 274 bp ID NO: 1) Reverse: 5′CGGGAACTTGAACTGGAACTGAC3′ (SEQID NO: 2) Nestin Forward: 5′CAGCTGGCGCACCTCAAGATG3′ (SEQ ID 208 bpNO: 3) Reverse: 5′AGGGAAGTTGGGCTCAGGACTGG3′ (SEQ ID NO: 4) Oct-4Forward: 5′-CGTTCTCTTTGGAAAGGTGTTC (SEQ ID 320 bp NO: 5) Reverse:3′-ACACTCGGACCACGTCTTTC (SEQ ID NO: 6) beta-actin Forward:5′-CGCACCACTGGCATTGTCAT-3′ (SEQ ID 200 bp NO: 7) Reverse:5′-TTCTCCTTGATGTCACGCAC-3′ (SEQ ID NO: 8) beta-actin Forward:5′-TCACCACCACGGCCGAGCG-3′ (SEQ ID 291 bp NO: 9) Reverse:5′-TCTCCTTCTGCATCCTGTCG-3′ (SEQ ID NO: 10) CD-34 Forward:5′-TGAAGCCTAGCCTGTCACCT-3′ (SEQ ID 200 bp NO: 11) Reverse:5′-CGCACAGCTGGAGGTCTTAT-3′ (SEQ ID NO: 12) FLK-1 Forward:5′-GGTATTGGCAGTTGGAGGAA-3′ (SEQ ID 199 bp NO: 13) Reverse:5′-ACATTTGCCGCTTGGATAAC-3′ (SEQ ID NO: 14) AC-133 Forward:5′-CAGTCTGACCAGCGTGAAAA-3′ (SEQ ID 200 bp NO: 15) Reverse:5′-GGCCATCCAAATCTGTCCTA-3′ (SEQ ID NO: 16) Hnf-3α Forward:5′-GAGTTTACAGGCTTGTGGCA-3′ (SEQ ID 390 bp NO: 17) Reverse:5′-GAGGGCAATTCCTGAGGATT-3′ (SEQ ID NO: 18) AFP Forward:5′-CCATGTACATGAGCACTGTTG-3′ (SEQ ID 340 bp NO: 19) Reverse:5′-CTCCAATAACTCCTGCTATCC-3′ (SEQ ID NO: 20) transferin Forward:5′-CTGACCTCACCTGGGACAAT-3′ (SEQ ID 367 bp NO: 21) Reverse:5′-CCATCAAGGCACAGCAACTC-3′ (SEQ ID NO: 22) Amylase Forward:5′-GCTGGGCTCAGTATTCCCCAAATAC-3′ 490 bp (SEQ ID NO: 23) Reverse:5′-GACGACAATCTCTGACCTGAGTAGC-3′ (SEQ ID NO: 24) α1 anti Forward:5′-AGACCCTTTGAAGTCAAGGACACCG-3′ 360 bp trypsin (SEQ ID NO: 25) Reverse:5′-CCATTGCTGAAGACCTTAGTGATGC-3′ (SEQ ID NO: 26) Keratin Forward:5′-AGGAAATCATCTCAGGAGGAAGGGC-3′ 780 bp (SEQ ID NO: 27) Reverse:5′-AAAGCACAGATCTTCGGGAGCTACC-3′ (SEQ ID NO: 28)

Amplification conditions were as follows: 94° C. for 4 min followed by40 cycles of 94° C. for 15 sec, 55° C. for 30 sec, 72° C. for 45 sec andextension at 72° C. for 7 min.

Products were analysed on a 1.5% or a 2% agarose gel and visualised byethidium bromide staining.

(c) Neuronal Differentiation Studies

In general, differentiation was induced by plating the spheres on anappropriate substrate (poly-D-lysine, 30-70 kDa, and laminin, Sigma)combined with the removal of growth factors.

Two protocols were most commonly used: In the first one, differentiationwas induced by plating the spheres on coverslips coated withpoly-D-lysine and laminin in the same serum free medium detailed abovewithout growth factors supplementation. The cells in the spheres wereallowed to spread and differentiate for 2-3 weeks and the medium waschanged every 3-5 days. In some of the experiments, starting from thesixth day after plating, the medium was supplemented with all transretinoic acid (Sigma, 10-6M).

In the second protocol, the spheres were plated on coverslips coatedwith poly-D-lysine and laminin in serum free growth medium supplementedwith growth factors. After 5-6 days the supplementation of growthfactors was withdrawn and all trans retinoic acid (Sigma, 10-6M) wasadded to the medium. The cells were further cultured for 1-2 weeks. Themedium was changed every 5 days.

(d) Characterization of Differentiated Neuronal Cells

Differentiated cells growing out from the spheres were analysed 2-3weeks after plating by indirect immunofluorescence for the expression ofthe following markers: 200 kDa neurofilament protein (4%paraformaldehyde fixation, mouse monoclonal antibody RT97 fromNovocastra, Newcastle, UK), 160 kDa neurofilament protein (methanolfixation without permeabilization, mouse monoclonal NN18 from Chemicon,Temecula, Calif.; 1:50) 68 kDa neurofilament protein (100% ethanol,Amersham, Amersham U.K), 70 kDa neurofilament protein (Chemicon; 1:100),MAP2 a,b (4% paraformaldehyde fixation, mouse monoclonal AP20 fromNeomarkers, Union City Calif.; 1:100), glutamate (1% paraformaldehydeand1% glutaraldehyde, rabbit antiserum from Sigma; 1:1000), synaptophysin(4% paraformaldehyde, mouse monoclonal SY38 from Dako; 1:50), tyrosinehydroxylase (4% paraformaldehyde, mouse monoclonal, Sigma), serotonin(Sigma; 1:1000), glutamic acid decarboxylase (GAD, 1% paraformaldehyde,1% glutaraldehyde, rabbit antiserum from Chemicon, Temecula, Calif.;1:200), GABA (4% paraformaldehyde, Sigma; 1:1000), β-tubulin (4%paraformaldehyde, mouse monoclonal TUB 2.1 from Sigma) and β-tubulin III(4% paraformaldehyde mouse monoclonal SDL.3D10 from Sigma; 1:150).

Differentiated cells were also analysed 2-3 weeks after plating byRT-PCR for the expression of β-actin, glutamic acid decarboxylase(primers, Vescovi et al., 1999) GABA_(A) receptor subunit α2 (primers,Neelands et al., 1998), neuron-specific enolase, neurofilament mediumsize chain (NF-M) (primers, Kukekov et al 1999). mRNA preparation andthe RT-PCR reaction were carried out as described above.

(e) Glial Differentiation Studies.

At the time of routine passage spheres were subcultured into serum freemedium (as detailed above) supplemented with platelet derived growthfactor (recombinant human PDGF-AA, Peprotech Inc 20 ng/ml) and bFGF(Gibco, 20 ng/ml). Fifty percent of the medium was replaced by freshmedium every 3 days. After culture for 6 days the spheres were plated oncoverslips coated with poly-D-lysine and laminin in the same serum freemedium without growth factors supplementation. The cells in the sphereswere allowed to spread and differentiate for 10-12 days and the mediumwas changed every 3-5 days. An alternative protocol was used in some ofthe experiments. In these experiments the spheres were cultured in serumfree medium (as detailed above) supplemented with PDGF-AA, (20 ng/ml)and bFGF (20 ng/ml) for three weeks. The spheres were then plated oncoverslips coated with poly-D-lysine and Fibronectin (Sigma, 5 mcg/mk).They were cultured for a week in the serum free medium supplemented withPDGF-AA, (20 ng/ml), bFGF (20 ng/ml) and T3 (30 nM). The growth factorswere then removed from the medium and the cells were further culturedfor another 1-2 weeks in the presence of T3 only. Fifty percent of themedium was replaced by fresh medium every 3 days. In an alternativeapproach, spheres that were propagated in the presence of EGF and bFGFwere plated on coverslips coated with poly-D-lysine and Fibronectin(Sigma, 5 mcg/mk). EGF was removed from the medium and the spheres werecultured in the presence of T3 (30 nM) and bFGF (20 ng/ml) for a week.The cells were then further cultured for another 3-4 weeks in thepresence of T3 and PDGF-AA (20 ng/ml).

Oligodendrocyte were identified by indirect immunofluorescence for theexpression of the marker O4. The cells were first incubated with theprimary (Anti oligodendrocyte marker O4, mouse monoclonal IgM fromChemicon Int. Inc. Temecula, Calif.; 1:10) and secondary FITC orrhodamine conjugated antibodies and were then fixed with 4%paraformaldehyde.

To demonstrate differentiation into astrocyte, spheres that have beenpropagated in the presence of b-FGF and EGF were plated on cover slipscoated with poly-D-lysine and fibronectin or laminin and furthercultured for 6 days in serum free medium without growth factorssupplementation.

Alternatively, spheres were propagated in the presence of PDGF-AA andbFGF for 6 weeks and were then plated on cover slips coated withpoly-D-lysine and fibronectin. They were allowed to spread into amonolayer in the presence of the above growth factors for a week. Thecells were then further cultured for another week in the presence ofeither T3 or the combination of T3 and PDGF-AA.

Following this protocols, differentiation into astrocytes wasdemonstrated by Indirect immunofluorescence for the expression of glialfibrillary acidic protein (GFAP) (4% paraformaldehyde fixation, rabbitanti cow from Dako; 1:50)

Differentiation into astrocyte and oligodendrocyte cells was alsoconfirmed at the mRNA level. To induce differentiation to theselineages, spheres were plated on poly-D-lysine and fibronectin andcultured for 2 weeks in the serum free medium supplemented with EGF,bFGF and PDGF-AA. The differentiating spheres were then further culturedtwo weeks without growth factors and in the presence of T3. RT-PCR wasused as above to demonstrate the expression of GFAP and the plp gene.GFAP transcripts were assayed using the following primers:5′-TCATCGCTCAGGAGGTCCTT-3′ (SEQ ID NO:29) (forward) and5′-CTGTTGCCAGAGATGGAGGTT-3′ (SEQ ID NO:30) (reverse), band size 383 bp.The primers for the analysis of plp gene expression were5′-CCATGCCTTCCAGTATGTCATC-3′ (SEQ ID NO:31) (forward) and5′-GTGGTCCAGGTGTTGAAGTAAATGT-3′ (SEQ ID NO:32) (reverse). The plp geneencodes the proteolipid protein and its alternatively spliced productDM-20 which are major proteins of brain myelin. The expected band sizefor plp is 354 bp and for DM-20 is 249 bp (Kukekov et al., 1999). As acontrol for mRNA quality, beta-actin transcripts were assayed using thesame primers as above. Products were analysed on a 2% agarose gel andvisualised by ethidium bromide staining.

(f) Transplantation Studies.

Spheres pre-labeled by the addition of 50 μM BrdU (Sigma) to the culturemedium for 10 days, were dissaggregated into small clumps eithermechanically or by enzymatic (papain, Wortinington Biochemical Co, NJ)digestion that was followed by tituration. Approximately 50,000 cells(in 2 μl PBS) were injected into the lateral ventricles of newborn(first day after birth) mice (Sabra mice, Harlan, Jerusalem) and rats(Sabra) by using a micro-glass pipette (300 micron outer diameter)connected to a micro-injector (Narishige, Japan). The glass pipette wascovered by a plastic sleeve that limited the depth of penetration intothe host nervous system. At 4-6 weeks of age, recipients wereanesthetized and perfused with 4% paraformaldehyde in PBS.

Detection and Characterization of Donor Human Neural Progenitors InVivo.

Serial 7-micrometer frozen sections were examined by immunostainingafter post fixation with acetone or with 4% paraformaldehyde andhistologically after hematoxylin and eosin staining. The transplantedcells were detected by immunuostaining with antibodies for BrdU (Dako,1:20, following specific instructions by the manufacturer), anti humanspecific ribonuclear protein antibody (Chemicon; 1:20) and anti humanspecific mitochondrial antibody (Chemicon; 1:20). BrdU antibody wasdetected by using the peroxidase-conjugated Vectastain kit (VectorBurlingame, Calif.), developed with Diaminobenzadine (DAB) or by usinggoat anti mouse IgG conjugated to Alexa 488 (Jackson; 1:100). Antiribonuclear protein and mitochondrial antibodies were detected with goatanti mouse IgM conjugated to Cy5 and goat anti mouse IgG conjugated toAlexa488, respectively (Jackson; 1:100). Glial cell type identity oftransplanted cells was established by double staining for BrdU and GFAP(Dako; 1:100) for astrocytes, CNPase (Sigma; 1:100) and NG2 (Chemicon;1:100) for the oligodendrocyte lineage. Neurons were detected byimmunostaining with human specific anti neurofilament light chain(Chemicon; 1:100) and anti βIII-tubulin (antibody as detailed above;1:100). Goat anti rabbit conjugated to Cy5 (Jackson; 1:100) and goatanti mouse IgG conjugated to Alexa488 (Jackson; 1:100) were used fordetection of primary antibodies. In cases where the lineage specificantibody was a mouse monoclonal IgG, double labeling with BrdU wasperformed sequentially, by first completing immunohistochemistry forBrdU and then performing immunofluorescent staining for the lineagemarker (i.e. CNPase or βIII-tubulin). Images were taken by a confocalmicroscope (Zeiss), using channels for Alexa488 fluorescence, Cy5fluorescence and Nomarsky optics.

EXAMPLES Example 1 Derivation of Cell Lines HES-1 and HES-2

The outer trophectoderm layer was removed from four blastocysts byimmunosurgery to isolate inner cell masses (ICM), which were then platedonto a feeder layer of mouse embryo fibroblasts (FIG. 1A). Withinseveral days, groups of small, tightly packed cells had begun toproliferate from two of the four ICM. The small cells were mechanicallydissociated from outgrowths of differentiated cells, and followingreplating they gave rise to flat colonies of cells with themorphological appearance of human EC or primate ES cells (FIG. 1B, Cstem cell colonies). These colonies were further propagated bymechanically disaggregation to clumps which were replated onto freshfeeder cell layers. Growth from small clumps of cells (<10 cells) wasnot possible under the conditions of these cultures. Spontaneousdifferentiation, often yielding cells with the morphological appearanceof early endoderm, was frequently observed during routine passage of thecells (FIG. 1D). Differentiation occurred rapidly if the cells weredeprived of a feeder layer, even in the presence of LIF (FIG. 1E). WhileLIF was used during the early phases of the establishment of the celllines, it was subsequently found to have no effect on the growth ordifferentiation of established cultures (not shown). Cell line HES-1 hasbeen grown for 60 passages in vitro and HES-2 for 40 passages,corresponding to a minimum of approximately 360 and 90240 populationdoublings respectively, based on the average increase in colony sizeduring routine passage, and both cell lines still consist mainly ofcells with the morphology of ES cells. Both cell lines have beensuccessfully recovered from cryopreservation.

Example 2 Marker Expression and Karyotype of the Human ES Cells

Marker and karyotype analysis were performed on HES-1 at passage levels5-7, 14-18, 24-26 and 44-46, and on HES-2 at passage levels 6-8. EScells contained alkaline phosphatase activity (FIG. 2A).Immunophenotyping of the ES cells was carried out using a series ofantibodies which detect cell surface carbohydrates and associatedproteins found on human EC cells. The ES cells reacted positively inindirect immunofluorescence assays with antibodies against the SSEA-4and TRA 1-60 carbohydrate epitopes, and the staining patterns weresimilar to those observed in human EC cells (FIG. 2B, C). ES cells alsoreacted with monoclonal antibody GCTM-2, which detects an epitope on theprotein core of a keratan sulphate/chondroitin sulphate pericellularmatrix proteoglycan found in human EC cells (FIG. 2D). Like human ECcells, human ES cells did not express SSEA-1, a marker for mouse EScells. Both cell lines were karyotypically normal and both were derivedfrom female blastocysts.

Oct-4 is a POU domain transcription factor whose expression is limitedin the mouse to pluripotent cells, and recent results show directly thatzygotic expression of Oct-4 is essential for establishment of thepluripotent stem cell population of the inner cell mass. Oct-4 is alsoexpressed in human EC cells and its expression is down regulated whenthese cells differentiate. Using RT-PCR to carry out mRNA analysis onisolated colonies consisting mainly of stem cells, we showed that humanES cells also express Oct-4 (FIG. 3, lanes 2-4). The PCR product wascloned and sequenced and shown to be identical to human Oct-4 (notshown).

Example 3 Differentiation of Human ES Cells In Vitro

Both cell lines underwent spontaneous differentiation under standardculture conditions, but the process of spontaneous differentiation couldbe accelerated by suboptimal culture conditions. Cultivation to highdensity for extended periods (4-7 weeks) without replacement of a feederlayer promoted differentiation of human ES cells. In high densitycultures, expression of the stem cell marker Oct-4 was eitherundetectable or strongly down regulated relative to the levels of thehousekeeping gene beta actin (FIG. 3, lanes 5-7). Alphafetoprotein andhuman chorionic gonadotrophin were readily detected by immunoassay inthe supernatants of cultures grown to high density. Alphafetoprotein isa characteristic product of endoderm cells and may reflect eitherextraembryonic or embryonic endodermal differentiation; the levelsobserved (1210-5806 ng/ml) are indicative of extensive endoderm present.Human chorionic gonadotrophin secretion is characteristic oftrophoblastic differentiation; the levels observed (6.4-54.6 IU/Litre)are consistent with a modest amount of differentiation along thislineage.

After prolonged cultivation at high density, multicellular aggregates orvesicular structures formed above the plane of the monolayer, and amongthese structures clusters of cells or single cells with elongatedprocesses which extended out from their cell bodies, forming networks asthey contacted other cells (FIG. 1F) were observed. The cells and theprocesses stained positively with antibodies against neurofilamentproteins and the neural cell adhesion molecule (FIGS. 2E and F).Contracting muscle was seen infrequently in the cultures. Whilecontracting muscle was a rare finding, bundles of cells which werestained positively with antibodies directed against muscle specificforms of actin, and less commonly cells containing desmin intermediatefilaments (FIGS. 2G and H) were often observed. In these high densitycultures, there was no consistent pattern of structural organisationsuggestive of the formation of embryoid bodies similar to those formedin mouse ES cell aggregates or arising sporadically in marmoset ES cellcultures.

Example 4 Differentiation of Human ES Cells in Xenografts

When HES-1 or HES-2 colonies of either early passage level (6; HES 1 and2) or late passage level (HES-1, 14 and 27) were inoculated beneath thetestis capsule of SCID mice, testicular lesions developed and werepalpable from about 5 weeks after inoculation. All mice developedtumours, and in most cases both testis were affected. Upon autopsylesions consisting of cystic masses filled with pale fluid and areas ofsolid tissue were observed. There was no gross evidence of metastaticspread to other sites within the peritoneal cavity. Histologicalexamination revealed that the lesion had displaced the normal testis andcontained solid areas of teratoma. Embryonal carcinoma was not observedin any lesion. All teratomas contained tissue representative of allthree germ layers. Differentiated tissues seen included cartilage,squamous epithelium, primitive neuroectoderm, ganglionic structures,muscle, bone, and glandular epithelium (FIG. 4). Embryoid bodies werenot observed in the xenografts.

Example 5 Development, Propagation and Characterisation of Human ESCells Derived Neural Progenitor Cells

a) Derivation of Neural Progenitor Cells from Human ES Cells.

Colonies of undifferentiated ES cells from the cell lines HES-1 andHES-2 were continuously cultured on mouse embryonic fibroblasts feederlayer for 2-3 weeks. At one week after passage, some spontaneousdifferentiation was usually identified by changes in cell morphology inthe center of the colonies. The process of differentiation included atthis early stage the neuroectodermal lineage as evident by theexpression of early neural markers such as nestin and PAX-6 (FIG. 19).During the next two weeks of culture, the process of differentiation wasmarkedly accelerated mainly in the center of the colonies and cells withshort processes that expressed the early neuroectodermal marker N-CAMcould be demonstrated. It appeared that the N-CAM positive cells weregrowing out from adjacent distinct areas that were comprised of smallpiled, tightly packed cells that did not express GCTM-2, a marker ofundifferentiated ES cells or the early neuroectodermal marker N-CAM(data not shown). These distinct areas had a typical uniform white grayopaque appearance under dark field stereo-microscopy (FIG. 26). Theseareas could be identified in the colonies of both cell lines from thesecond week after passage, and they became more defined from neighboringareas of the colony during the third week of culture (FIG. 26). The sizeand demarcation of these areas was enhanced if the serum containing EScell culture medium was replaced after a week or preferably after twoweeks from passage with serum free medium supplemented with EGF (20ng/ml) and FGF (20 ng/ml). The areas were large and well demarcatedsufficiently to allow mechanical removal of clumps of cells by amicropipette in 54% of the colonies cultured in serum containing medium(67/124, HES-1). Clumps were removed from differentiating colonies ofHES-1 and HES-2 and were transferred to serum free medium supplementedwith basic fibroblast growth factor (bFGF) and epidermal growth factor(EGF). At the time of isolation, the clumps were comprised mostly of alayer of the small tightly packed cells (about 100-300 cells/clump), ontop of some loosely attached larger cells, It was possible to removethese larger cells mechanically or by enzymatic digestion. Within anhour the clumps started to change their shape toward spheres and after24 hours all the clumps turned into round spheres (FIG. 5 a).

During the initial two weeks in culture, some cell death was observed.After 7-10 days in culture, gradual increase in the size of the majorityof the spheres was evident and most of the spheres were still floatingor loosely attached to the dish while a minority attached and started tospread. A detailed analysis of marker expression and the growth anddifferentiation potential of the cells within the spheres was conductedin three preparations of spheres that were separately derived with thisapproach.

In an alternative approach, somatic differentiation of ES cells intospheres of progenitor cells was induced by transferring clumps ofundifferentiated ES cells into serum free medium supplemented with basicfibroblast growth factor (bFGF) and epidermal growth factor (EGF).Within 24 hours the clumps have turned into spheres. Some of thesespheres were round and some had an irregular shape. After 72 hours inserum free medium 42% (10/24) of the spheres had a round symmetricalappearance (FIG. 9) and after 12 days 62.5% (15/24). Significant growthwas observed in the majority of the spheres during this early culture.It was possible to measure and calculate the average volume of the roundfloating spheres and it increased by 64% (mean growth of 15 spheres)between days 5 and 12. Two preparations of spheres that were separatelyderived with this approach were further propagated and characterized.

b) In Vitro Propagation of Spheres of Progenitor Cells

After 7-10 days in culture, floating or loosely attached spheres with adiameter of >0.5 mm were sub-cultured by mechanical dissection into 4pieces, which were re-plated in fresh pre-equilibrated medium. Thespheres were cultivated in this manner during a five to six month period(15 passages). Although some of the spheres had an irregular shape atthe initial phase of culture, the rate of round symmetrical spheresincreased along propagation. In addition, while at early passage levelsthe appearance (under a stereo-microscope) of the inner part of thespheres was irregular, it gradually turned to be uniform at moreadvanced passage levels. By passage level five (five-six weeks afterderivation) all spheres had a round symmetrical shape and a uniformappearance.

Proliferation of the cells was evaluated by determining the increase inthe number of spheres with each passage as well as measuring theincrease in the volume of the spheres along time. In general, the growthrate of spheres that were generated either from undifferentiated or fromdifferentiating ES cells had a similar pattern characterized by a moreexcessive growth during the first 5-6 passages. The number of spheresincreased by 126%+54% (Mean+SD, sum of results from 3 cell lines) ateach passage (performed every 7 days) during the first 5 passages. Thegrowth of the number of spheres with each passage was then reduced to10-50% per week. This growth rate was maintained for prolonged periods(4 months) (mean data from 3 cell lines). The mean volume of spheresgenerated either from differentiating ES cells or directly fromundifferentiated cells also increased by similar rates (FIGS. 16 and17). A relatively rapid growth rate was observed during the first 5-6weeks after derivation with a population doubling time of approximately4.7 days. It was followed by a 10-16 week period of slow and stable cellgrowth with a population doubling time of approximately two and a halfweeks. At this point the spheres ceased to grow and their volume wasstable or declined (FIG. 31).

Dis-aggregation of the spheres by using trypsin digestion could beineffective in particular when the spheres were cultivated for prolongedperiods, however it was possible to dis-aggregate them into a singlecell suspension mechanically following enzymatic digestion with papain.A linear correlation was found between the volume of spheres and thenumber of cells within the spheres at various passage levels (5-15 weeksafter derivation) indicating the validity of monitoring the increment ofsphere volume as an indirect indicator of cell proliferation. Thecoefficients that define the regression line of this correlation weresimilar in spheres that were derived from differentiating orundifferentiated ES cells. Most of the cells (>90%) were viablefollowing the dissagregation procedure (FIG. 10, FIG. 18).

Given the growth rate of the spheres with each passage and the number ofcells (20,000, FIG. 10, 18) per averaged size sphere (0.1 mm³ based onthe mean diameter±S.D. of 24 spheres 7 days after passage 5, 0.59±0.14mm), it was calculated that 10 clumps of ES cells may generate within 10passages 2500 spheres containing 50×10⁶ cells. A cumulative growth curveis presented in FIG. 31 to illustrate this potential for significantexpansion.

Spheres that were cultured in the serum free growth medium (supplementedwith growth factors) for prolonged periods (3 weeks) without passage,tended to attach to the tissue culture plastic and gradually spread as amonolayer of cells. The cells in the monolayer had a uniform appearanceof neural progenitors and a high mitotic activity was evident (FIG. 7).

It was possible to recover the spheres from cryopreservation.

c) Characterization of the Progenitor Cells within the Spheres.

Cells in the spheres that were produced either from differentiating EScell colonies or directly from undifferentiated ES cells expressedmarkers of primitive neuroectoderm and neural progenitor cells, such aspolysialylated N-CAM (FIG. 5 b, 11,12), A2B5, the intermediate filamentproteins nestin (immunostaining, FIG. 5 c and 13; RT-PCR, FIG. 3 b andFIG. 19) and vimentin (FIGS. 5 d and 15), and the transcription factorPax-6 (FIG. 3 b and FIG. 19). The expression of these markers wasmaintained along prolonged cultivation (18 weeks). The transcriptionalfactor oct4 was not expressed by the cells in the spheres indicatingthat undifferentiated human ES cells were not present within the spheres(FIG. 19).

To evaluate the proportion of cells in the spheres that expressed N-CAM,A2B5, nestin and vimentin, spheres that were cultivated at least 6 weeks(and up to 18 weeks) were disaggregated to single cell suspension. Theresulting single cells were plated on substrate in growth medium. Twentyfour hours after plating, an average of 99±1.6% (n=11 experiments) and95.5% (95.7%-96.7%, n=6) of the cells from spheres generated formdifferentiating (three progenitor cell lines) and undifferentiated EScells (two progenitor cell lines) respectively were decorated with theantibody against N-CAM. The average proportion of cells that werepositively stained for nestin, A2B5, and vimentin was 97±2.3% (n=10experiments) 89.5% (n=2 experiments) and 67±16.8% (n=9 experiments)respectively in spheres that were established from differentiatingcolonies. Nestin and vimentin were expressed by 66.8% (48.5%-100%, n=5)and 58% (41.6%-76.5%, n=5) of the cells that originated from spheresthat were generated from undifferentiated cells. These proportions werestable during prolonged cultivation (18 weeks).

The high proportion of cells that expressed N-CAM indicate that thespheres from both sources were comprised of a highly enrichedpreparation of neural progenitor cells. An extremely high proportion ofcells from spheres that were derived from differentiating ES coloniesalso expressed the neural progenitor markers nestin and A2B5. Theproportion of cells expressing nestin was less extensive in spheres thatoriginated from undifferentiated ES cells. The high proportion of cellsthat expressed these markers was stable along prolonged cultivation.

To determine whether cells from other lineages were present within thespheres, the expression of markers of endoderm, epidermis and mesodermwas examined by RT-PCR and immunohistochemistry.

There was no evidence for the expression of markers of theextraembryonic endodermal lineage (HNF-3α, AFP, RT-PCR, FIG. 24) bycells of spheres that were derived by either methods. Moreover, theexpression of markers of the endodermal lineage was also not detected inspheres that were derived from differentiating colonies and that wereinduced to differentiate by plating on an appropriate substrate andculturing in the absence of growth factors for 4 weeks (HNF-3α, AFP,were evaluated by RT-PCR; LMW cytokeratin and laminin were_evaluated byimmunohistochemistry). ES cell colonies that were induced todifferentiate by prolonged culture (3-4 weeks) expressed all of theabove markers and served as positive controls.

However, expression of markers of mesodermal precursors (FLK-1 andCD-34) was demonstrated in the spheres that were produced by eithermethod (FIG. 24). In addition, transcripts of markers of embryonicendoderm (α1 anti trypsin, transferrin and amylase), epidermis (keratin)and the hematopoietic lineage (Ac-133) were demonstrated in spheres thatwere derived from differentiating ES colonies (FIG. 32).

It may be concluded that the spheres were comprised of a highly enrichedpopulation of neural progenitors (>95%) and probably no cells from theextraembryonic endodermal lineage. The expression of transcripts ofnon-neural markers may indicate the presence of a minute population ofcells from other lineages within the spheres. Alternatively it may bethat the primitive neural precursors within the spheres express thesemarkers. The expression of the hematopoietic marker AC-133 (Uchida etal., 2000) that was recently demonstrated in neural stem cells derivedfrom human fetal brains support this possibility. In addition,expression of non-neural markers by the neural progenitors may be inline with the recently reported broad potential of neural stem cells totrans-differentiate into a variety of tissues (Bjornson et al., 1999;Clarke et al., 2000).

(d) In Vitro Neuronal Differentiation

When plated on poly-D-lysine and laminin, spheres that were producedeither from differentiating ES cell colonies or from undifferentiated EScells attached, and differentiated cells grew out onto the monolayerfrom them.

When the bFGF and EGF were removed at the time of plating,differentiating cells gradually spread from them in a radial fashion(FIG. 6 a, FIG. 33) If the growth factors were removed only after 1-2weeks, a more extensive spreading of cells with processes, which formeda monolayer was evident (FIG. 8). Two to three weeks after plating, thedifferentiated cells originating from spheres derived by either methodsdisplayed morphology and expression of structural markers characteristicof neurons, such as β-tubulin (FIG. 6 h), β-tubulin III (FIG. 27 c, the200 kDa neurofilament (FIG. 6 b) and 68 kDA neurofilament proteins andneuron specific enolase (NSE, FIG. 34). Moreover, differentiated cellsoriginating from spheres derived by either methods expressed markers ofmature neurons such as the 160 kDa neurofilament protein (FIG. 6 c, FIG.27 a, FIG. 34), Map-2a,b (FIG. 6 d, FIG. 27 b) and synaptophysin (FIG.6F). Furthermore, the cultures contained cells which synthesisedglutamate (FIG. 6 e), serotonin (FIG. 35 a), GABA (FIG. 35 b), expressedthe rate limiting enzyme in GABA biosynthesis (glutamic aciddecarboxylase, FIGS. 3 c and 6 g), expressed the enzyme tyrosinehydroxylase (FIG. 28) and receptor subunits characteristic of GABAminergic neurons (GABAα2, FIG. 3 d).

e) In Vitro Glial Differentiation

Differentiation into both astrocyte cells and oligodendrocyte cells wasobserved with spheres that were produced either from differentiating EScells or from undifferentiated cells.

While differentiation at a low scale toward glial cells was observedupon withdrawal of growth factors and plating on poly-D-lysine andlaminin, various protocols were developed to enhance the differentiationtoward this lineage. These protocols were all based on plating theneural progenitor cells on poly-D-lysin and fibronectin, whichsignificantly enhanced the differentiation toward glial cells, andsupplementation of the medium with PDGF-AA that promotes glialprogenitor cell proliferation and T3 that induces maturation ofoligodendrocytes precursors.

Differentiation into the astrocyte glial lineage was demonstrated byindirect immunofluorescent staining for GFAP. Few positive cells wereoccasionally demonstrated when differentiation was induced by withdrawalof growth factors and plating on poly-D-lysin and laminin. However,differentiation into astrocytes was significantly enhanced when thespheres were allowed to differentiate on poly-D-lysin and fibronectin.Moreover, differentiation into astrocytes was highly abundant after thefollowing protocol. The spheres were first propagated six weeks in thepresence of PDGF-AA and bFGF and were then plated on poly-D-lysine andfibronectin. They were allowed to spread for a week into a monolayer inthe presence of the above growth factors. The differentiating cells werethen further cultured for another week in the presence of T3 and PDGF-AAfollowed by another 1-2 weeks of culture either with T3 or thecombination of T3 and PDGF-AA (FIG. 20).

To promote differentiation towards oligodendrocyte, spheres wereinitially cultured for 6 days in serum free medium supplemented withPDGF-AA (20 ng/ml) and bFGF (20 ng/ml) and were then plated oncoverslips coated with poly-D-lysine and laminin in the same mediumwithout growth factors supplementation. The cells in the spheres wereallowed to spread and differentiate for 10-12 days. Small cellsdecorated with the antibody O4 could be demonstrated at that timeindicating differentiation into oligodendrocyte progenitors.

It was also possible to promote the differentiation into oligodendrocyteprogenitors by incubation of the spheres in the presence of PDGF andbasic FGF for 3 weeks followed by plating on poly lysine and fibronectinand culture for a week in the presence of the growth factors and T3followed by 1-2 weeks culture in the presence of T3 without growthfactors supplementation (FIG. 14). Alternatively, spheres that werepropagated in the presence of bFGF and EGF were plated on poly lysineand fibronectin and cultured for a week in the presence of bFGF and T3.The cells were then further cultured in the presence of PDGF and T3 for3-4 weeks.

Differentiation into astrocyte and oligodendrocyte cells was furtherconfirmed at the mRNA level. Spheres were plated on poly-D-lysine andfibronectin and cultured for 2 weeks in the serum free mediumsupplemented with EGF, bFGF and PDGF-AA. The differentiating sphereswere then further cultured two weeks without growth factors in thepresence of T3. The expression of GFAP was demonstrated by RT-PCRindicating and confirming the presence of astrocyte cells (FIG. 25, FIG.34). The expression of myelin basic protein (MBP) and the plp gene wasused as markers of differentiation into oligodendrocyte cells. The plpgene encodes the proteolipid protein and its alternatively splicedproduct DM-20, which are major proteins of brain myelin.

RT-PCR analysis of the differentiated spheres demonstrated MBP and bothdm-20 and plp transcripts indicating that differentiation intooligodendrocyte has occurred (FIG. 25, FIG. 34).

f) Transplantation of Neural Spheres.

To explore the developmental potential of the human ES-derived neuralprogenitors in vivo, and to reveal whether the human precursors canrespond to positional cues, migrate along established host brain tracts,differentiate into neural cell types according to a given region's stageof development and participate in the development and histogenesis of aliving host, dis-aggregated spheres were implanted into the lateralcerebral ventricles of newborn mice. Transplantation was performed 9-15weeks after derivation of the neural spheres. Prior to transplantation,the neural progenitors were labeled with BrdU to facilitate theiridentification in the host brain. Histological and immunochemicalevaluation of serial brain sections was performed mainly 4-6 weeks aftertransplantation. The transplanted cells were identified by theirimmunoreactivity with anti BrdU antibodies (FIGS. 21 and 36E). NumerousBrdU+ cells were found in 9 out of 14 transplanted animals andsuccessful engraftment was documented with donor cells from the threeneural progenitor clones. The human identity of cells that weredecorated with anti BrdU was confirmed by double labeling demonstratingimmunoreactivity with both anti BrdU and anti human specificribonucleoprotein antibodies (FIG. 37B). The identity of thetransplanted human cells was also confirmed by immunofluorescentstaining with a human-specific anti-mitochondrial antibody (FIG. 37A).

Transplanted newborns that were examined during the first postnatal weekexhibited clusters of donor cells lining the ventricular wall (FIG.36A). At 4-6 weeks following implantation, human cells had left theventricles and migrated in large numbers mainly as individual cells intothe host brain parenchyma. The human cells demonstrated a widespreaddistribution in various regions of the host brain includingperiventricular areas (FIG. 22), the entire length of the corpuscallosum, fimbria, internal capsule, as well as into diencephalic tissuearound the 3rd ventricle (FIG. 36B, D). Transplanted human cells alsomigrated anteriorily from the subventricular zone along the rostralmigratory stream (FIG. 23, FIG. 36C) and populated the olfactory bulb,indicating their potential to respond to local cues and migrate alongestablished host brain tracts. In addition, BrdU+ cells were found inthe dentate gyrus (not shown), where post-natal neuronogenesis is knownto occur as well.

Differentiation in vivo into the three fundamental neural lineages wasdemonstrated by immunochemical studies using anti human cell typespecific antibodies or double labeling experiments with both anti BrdUand anti neural cell type specific antibodies. Glial differentiation ofthe transplanted cells was abundant in the periventricular areas thatconsist of white matter tracks where glial differentiation in the postnatal period is predominant. Double labeling immunochemical studies forBrdU and GFAP demonstrated cells that were decorated by both antibodiesindicating in-vivo differentiation into astrocytes (FIG. 29, FIG. 37C).Transplanted cells that have differentiated into the oligodendrogliallineage and were reactive with anti BrdU and anti NG-2 (a marker ofoligodendrocyte progenitors) or anti 2′,3′-cyclic nucleotide3′-phosphodiesterase (CNPase), were also demonstrated in these areas(FIG. 30 and FIG. 37D and E).

Neuronal differentiation of the human transplanted cells wasspecifically demonstrated in the olfactory bulb, a region whereneuronogenesis occurs after birth. Neuronal processes of transplantedcells were also detected by a human specific anti light chainneurofilament antibody in the fimbria (FIG. 37F and G).

There was no histological evidence of tumor formation in any of therecipient animals.

Our data demonstrated that neural progenitors that were derived fromhuman ES cells in vitro could respond appropriately to normaldevelopmental cues in vivo. Following transplantation to the cerebralventricles of newborn mice, the donor cells migrated in large numbersinto the host brain parenchyma and demonstrated widespread distribution.Migration was not random and the transplanted human progenitors followedestablished brain pathways as demonstrated by their migration along theRMS, suggesting their responsiveness to host's signals. The human ESderived neural progenitors differentiated in vivo into neurons,astrocyte and oligodendrocyte cells. Differentiation into neurons wasdemonstrated only in the olfactory bulb where host differentiation intothis lineage occurs in the postnatal period, whereas differentiationinto astroglia and oligodendroglia was demonstrated in subcortical areaswhere gliogenesis predominates and neurogenesis has ended. These datademonstrate that cell fate was determined in a region specific mannerand according to a given region's stage of development. It should benoted that the ability to integrate and participate in host brainhistogenesis was maintained after prolonged proliferation of the ESderived neural progenitors in vitro.

Example 6 Cryo-Preservation of Human ES Cells

Attempts to cryo-preserve human ES cells by using conventional slowfreezing protocols were associated with a very poor outcome afterthawing. Since ES cells are derived from the blastocyst and retain theirembryonic properties in culture, we have postulated thatcryopreservation by using a method which is efficient for embryos may bebeneficial. Early passage clumps of human ES cells were frozen by usingthe open pulled straw (OPS) vitrification method which was recentlyshown to be highly efficient for the cryopreservation of bovineblastocysts (Vatja et al. 1998). Both cell lines were successfullythawed and further propagated for prolonged periods. The outcome of thevitrification procedure was further studied on cell line HES-1, andrecovery of viable cells with this procedure was found to be highlyefficient. All clumps (n=25) survived the procedure and attached andgrew after thawing. Vitrification was associated with some cell death asevidenced by the reduced size of colonies originating from vitrifiedclumps two days after thawing in comparison to colonies fromnon-vitrified control clumps. However, two days in culture weresufficient to overcome this cell deficit, and 9 days after plating thesize of colonies from frozen-thawed clumps exceeded that of controlcolonies at 7 days. Vitrification did not induce differentiation afterthawing. Thawed cells retained a normal karyotype and the expression ofprimate stem cell markers, and formed teratomas in SCID mice.

Finally it is to be understood that various other modifications and/oralterations may be made without departing from the spirit of the presentinvention as outlined herein.

What is claimed is:
 1. An enriched population of neural progenitorcells, wherein at least 95% of the cells of the population expressN-CAM.
 2. The enriched population of neural progenitor cells of claim 1,generated by continuous culturing undifferentiated human embryonic stem(ES) cells for 2-3 weeks on a fibroblast feeder layer to generatecolonies and selecting from said colonies, small piled, tightly packed,differentiated cells.
 3. The enriched population of neural progenitorcells of claim 1, generated by culturing undifferentiated ES cells inthe presence of serum free medium supplemented with growth factors whichinclude epidermal growth factor (EGF) and basic fibroblast growth factor(bFGF).
 4. The enriched population of neural progenitor cells of claim1, wherein at least 97% of said cell express nestin.
 5. The enrichedpopulation of neural progenitor cells of claim 1, wherein at least 89%of said cell express A2B5.
 6. The enriched population of neuralprogenitor cells of claim 1, wherein at least 67% of said cell expressvimentin.
 7. The enriched population of neural progenitor cells of claim1, wherein at least 66% of said cell express nestin.
 8. The enrichedpopulation of neural progenitor cells of claim 1, wherein at least 58%of said cell express vimentin.